Griffithsin, glycosylation-resistant griffithsin, and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of use

ABSTRACT

An isolated and purified nucleic acid molecule that encodes a polypeptide comprising at least eight contiguous amino acids of SEQ ID NO: 3, wherein the at least eight contiguous amino acids have anti-viral activity, as well as an isolated and purified nucleic acid molecule that encodes a polypeptide comprising at least eight contiguous amino acids of SEQ ID NO: 3, wherein the at least eight contiguous amino acids have anti-viral activity, and, when the at least eight contiguous amino acids comprise amino acids 1-121 of SEQ ID NO: 3, the at least eight contiguous amino acids have been rendered glycosylation-resistant, a vector comprising such an isolated and purified nucleic acid molecule, a host cell comprising the nucleic acid molecule, optionally in the form of a vector, a method of producing an anti-viral polypeptide or conjugate thereof, the anti-viral polypeptide itself, a conjugate or fusion protein comprising the anti-viral polypeptide, and compositions comprising an effective amount of the anti-viral polypeptide or conjugate or fusion protein thereof. Further provided are methods of inhibiting prophylactically or therapeutically a viral infection of a host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 11/569,813, filed Dec. 12, 2006, which issued as U.S. Pat. No.7,884,178, which is the U.S. national phase of PCT/US05/18778, filed onMay 27, 2005, which claims the benefit of U.S. Provisional PatentApplication No. 60/576,056, filed Jun. 1, 2004, which is incorporated byreference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 8,345 Byte ASCII (Text) file named“707632ST25.TXT,” created on Feb. 3, 2011.

Applicants respectfully request entry into the specification of theSequence listing submitted herewith.

TECHNICAL FIELD OF THE INVENTION

The invention relates to an anti-viral polypeptide, aglycosylation-resistant anti-viral polypeptide, and related conjugates,compositions, nucleic acids, vectors, host cells, antibodies and methodsof production and use.

BACKGROUND OF THE INVENTION

The field of viral therapeutics has developed in response to the needfor agents effective against retroviruses, especially HIV. There aremany ways in which an agent can exhibit anti-retroviral activity (e.g.,see DeClercq, Adv. Virus Res., 42: 1-55 (1993); DeClercq, J. Acquir.Immun. Def. Synd., 4: 207-218 (1991); and Mitsuya et al., Science, 249:1533-1544 (1990). Nucleoside derivatives, such as AZT, which inhibit theviral reverse transcriptase, were among the first clinically activeagents available commercially for anti-HIV therapy. Although very usefulin some patients, the utility of AZT and related compounds is limited bytoxicity and insufficient therapeutic indices for fully adequatetherapy. Also, given the subsequent revelations about the true dynamicsof HIV infection (Coffin, Science, 267: 483-489 (1995); and Cohen,Science, 267: 179 (1995)), it has become increasingly apparent thatagents acting as early as possible in the viral replicative cycle areneeded to inhibit infection of newly produced, uninfected immune cellsgenerated in the body in response to the virus-induced killing ofinfected cells. Also, it is essential to neutralize or inhibit newinfectious virus produced by infected cells.

Effective means for preventing HIV infection also are needed as a globalpriority. Heterosexual transmission accounts for the majority of newcases of HIV infection each year. Current reports from the World HealthOrganization estimate that a total of more than 40 million people arenow infected with HIV. HIV prevention research has to date focusedpredominantly on vaccine development. However, no effective preventativeor therapeutic vaccine has been identified thus far. New approaches tovaccine development, as well as entirely different strategies and agentsfor preventing person-to-person transmission of HIV infection, areneeded. One approach showing great promise is the development and use oftopical microbicides. In this approach, a suitable antiviral agent isapplied directly at the potential site of virus exposure, e.g., thegenital mucosa in the case of HIV. A suitable antiviral agent is onewhich inactivates or inhibits infectivity of a virus upon contact of theantiviral agent with the virus. Suitable animal models are available fordemonstrating in vivo efficacy of such approaches for preventingtransmission of immunodeficiency viruses, such as HIV. For instance, theHIV-inactivating protein, cyanovirin-N, has been shown to inhibit thesexual transmission of a chimeric simian/human immunodeficiency virus(SHIV) infection in a primate model employing macaques exposed to thevirus vaginally or rectally (C-C Tsai et al., AIDS Res. Hum.Retroviruses, 19, 535-541 (2003) and C-C Tsai et al., AIDS Res. Hum.Retroviruses, 20, 11-18 (2004)).

Infection of people by influenza viruses is also a major cause ofpandemic illness, morbidity and mortality worldwide. The adverseeconomic consequences, as well as human suffering, are enormous.Available treatments for established infection by this virus are eitherminimally effective or ineffective; these treatments employamantatadine, rimantadine and neuraminidase inhibitors. Of these drugs,only the neuraminidase inhibitors are substantially active againstmultiple strains of influenza virus that commonly infect humans, yetthese drugs still have limited utility or efficacy against pandemicdisease.

Currently, the only effective preventative treatment against influenzaviral infection is vaccination. However, this, like the drug treatments,is severely limited by the propensity of influenza viruses to mutaterapidly by genetic exchange, resulting in the emergence of highlyresistant viral strains that rapidly infect and spread throughoutsusceptible populations. In fact, a vaccination strategy is onlyeffective from year-to-year if the potential pandemic strains can beidentified or predicted, and corresponding vaccines prepared andadministered early enough that the year's potential pandemic can beaborted or attenuated. Thus, new preventative and therapeuticinterventions and agents are urgently needed to combat influenzaviruses.

New agents with broad anti-influenza virus activity against diversestrains, clinical isolates and subtypes of influenza virus would behighly useful, since such agents would most likely remain active againstthe mutating virus. The two major types of influenza virus that infecthumans are influenza A and B, both of which cause severe acute illnessthat may include both respiratory and gastrointestinal distress, as wellas other serious pathological sequellae. An agent that hasanti-influenza virus activity against diverse strains and isolates ofboth influenza A and B, including recent clinical isolates thereof,would be particularly advantageous for use in prevention or treatment ofhosts susceptible to influenza virus infection.

The predominant mode of transmission of influenza viral infection isrespiratory, i.e., transmission via inhalation of virus-ladenaerosolized particles generated through coughing, sneezing, breathing,etc., of an influenza-infected individual. Transmission of infectiousinfluenza virions may also occur through contact (e.g., throughinadvertent hand-to-mouth contact, kissing, touching, etc.) with salivaor other bodily secretions of an infected individual. Thus, the primaryfirst points of contact of infectious influenza virions within asusceptible individual are the mucosal surfaces within the oropharyngealmucosa, and the mucosal surfaces within the upper and lower respiratorytracts. Not only do these sites comprise first points of virus contactfor initial infection of an individual, they are also the primary sitesfor production and exit (e.g., by coughing, sneezing, salivarytransmission, etc.) of bodily fluids containing infectious influenzaviral particles. Therefore, availability of a highly potentanti-influenza virus agent, having broad-spectrum activity againstdiverse strains and isolates of influenza viruses A and B, which couldbe applied or delivered topically to the aforementioned mucosal sites ofcontact and infection and transmission of infectious influenza viruses,would be highly advantageous for therapeutic and preventative inhibitionof influenza viral infection, either in susceptible uninfected orinfected hosts.

In this regard, new classes of anti-viral agents, to be used alone or incombination existing anti-viral agents, are needed for effectiveanti-viral therapy. New agents are also important for the prophylacticinhibition of viral infection. In both areas of need, the ideal newagent(s) would act as early as possible in the viral life cycle; be asvirus-specific as possible (i.e., attack a molecular target specific tothe virus but not the host); render the intact virus noninfectious;prevent the death or dysfunction of virus-infected cells; preventfurther production of virus from infected cells; prevent spread of virusinfection to uninfected cells; be highly potent and active against thebroadest possible range of strains and isolates of a given virus; beresistant to degradation under physiological and rigorous environmentalconditions; and be readily and inexpensively produced.

Accordingly, the invention provides a novel anti-viral polypeptide andrelated conjugates, nucleic acids, vectors, host cells and methods ofproduction and use. This and other advantages of the invention, as wellas additional inventive features, will become apparent from thedescription provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides, among other things, an isolated and purifiednucleic acid molecule that encodes a polypeptide comprising at leasteight contiguous amino acids of SEQ ID NO:3, wherein the at least eightcontiguous amino acids have anti-viral activity, optionally as part ofan encoded fusion protein. In this regard, the invention also providesan isolated and purified nucleic acid molecule that encodes apolypeptide comprising at least eight contiguous amino acids of SEQ IDNO:3, wherein the at least eight contiguous amino acids comprise aminoacids 1-121 of SEQ ID NO:3 which have been renderedglycosylation-resistant and wherein the at least eight contiguous aminoacids have antiviral activity, optionally as part of an encoded fusionprotein. Further provided are vectors comprising an aforementionedisolated and purified nucleic acid molecule and a host cell or organismcomprising such a vector.

Accordingly, the invention also provides a method of producing ananti-viral polypeptide, which method comprises expressing the nucleicacid molecule, optionally in the form of a vector, in a host cell ororganism. Thus, an anti-viral polypeptide comprising at least eightcontiguous amino acids of SEQ ID NO:3, wherein the at least eightcontiguous amino acids have anti-viral activity, and an antiviralpolypeptide comprising at least eight contiguous amino acids of SEQ IDNO:3, wherein the at least eight contiguous amino acids comprise aminoacids 1-121 of SEQ ID NO:3, which have been renderedglycosylation-resistant and wherein the at least eight contiguous aminoacids have antiviral activity, are also provided, as are conjugatescomprising an aforementioned anti-viral polypeptide and at least oneeffector component. Compositions comprising an effective amount of anaforementioned anti-viral polypeptide or anti-viral polypeptideconjugate are also provided.

The invention further provides a method of inhibiting prophylacticallyor therapeutically a viral infection of a host, specifically aretroviral infection of a host, such as an infection of a host with ahuman immunodeficiency virus (HIV), e.g., HIV-1 or HIV-2, or influenzavirus. The method comprises administering to the host an effectiveamount of an anti-viral polypeptide or anti-viral polypeptide conjugatecomprising at least eight contiguous amino acids of SEQ ID NO:3, whereinthe at least eight contiguous amino acids have anti-viral activity,whereupon the viral infection is inhibited.

Still further provided is a method of inhibiting a viral infection of ananimal comprising transforming host cells in vivo with a nucleic acidmolecule encoding an above-described polypeptide. Even still furtherprovided is a method of inhibiting a viral infection of an animalcomprising transforming host cells with a nucleic acid molecule encodingan above-described polypeptide and placing the transformed host cellsinto or onto the animal.

An antibody that binds griffithsin is provided as is a compositioncomprising same. Similarly, an anti-griffithsin antibody is provided asis a composition comprising same. A method of administering ananti-griffithsin antibody or a composition comprising same to a mammalso as to inhibit infection of the mammal with a virus is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram illustrating an anti-HIV bioassay-guided methodof isolating, purifying, and elucidating the amino acid sequence ofgriffithsin.

FIG. 2 is a flow diagram illustrating a method of synthesizing arecombinant griffithsin gene.

FIG. 3 is a flow diagram illustrating a method of expressing a syntheticgriffithsin gene encoding a His-tagged griffithsin polypeptide proteinand purification of the recombinant His-tagged griffithsin.

FIG. 4 a is a line graph illustrating the anti-HIV activity of nativegriffithsin, in terms of concentration of griffithsin (nM) (X-axis)versus % control (Y-axis). FIG. 4 b is a line graph illustrating theanti-HIV activity of recombinant, His-tagged griffithsin in terms ofconcentration of griffithsin (nM) (X-axis) versus % control (Y-axis).

FIG. 5 a is a bar graph comparing test proteins bound by griffithsin(Y-axis) and absorbance of the griffithsin-test protein complex at 405nm (X-axis). FIG. 5 b illustrates the concentration-dependent binding ofgriffithsin to glycosylated (•) or nonglycosylated (∘) gp120 bycomparing griffithsin (GRFT) concentration (pmol) and absorbance ofgriffithsin-gp120 complexes at 405 nm.

FIG. 6 is a flow diagram illustrating a method of producinganti-griffithsin antibodies.

FIG. 7 is the amino acid sequence of griffithsin polypeptide (SEQ IDNO:3) isolated and purified from Griffithsin sp.

FIG. 8 shows the nucleic acid (SEQ ID NO:1) sequence of recombinantgriffithsin.

FIG. 9 is the amino acid sequence of a recombinant griffithsinpolypeptide (SEQ ID NO:2).

FIG. 10 shows the nucleic acid sequence of a recombinant griffithsinpolypeptide comprising a His tag (SEQ ID NO:4).

FIG. 11 is the amino acid sequence of a recombinant griffithsinpolypeptide comprising a His tag (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The principal overall objective of the invention is to provide ananti-viral polypeptide and derivatives thereof, and broad uses thereof(e.g., medical and research uses), including prophylactic and/ortherapeutic applications against viruses. An initial observation, whichled to the invention, was anti-viral activity of certain extracts from amarine organism, namely Rhodophyte (Griffithsia sp.), originallycollected in the territorial waters of New Zealand. Low picomolarconcentrations of a protein isolated from the extracts, referred toherein as griffithsin, irreversibly inactivated human clinical isolatesof HIV. Its HIV molecular target is high mannose-comprisedoligosaccharide constituents of Env glycoproteins. Upon binding,griffithsin inhibits viral binding, fusion, and entry. Griffithsin alsotargets other viruses having oligosaccharide constituents similar toHIV, such as other retroviruses, e.g., FIV, SIV and HTLV, andnon-retroviruses, e.g., influenza, ebola, and measles.

Accordingly, the invention provides an isolated and purified anti-viralpolypeptide of SEQ ID NO:3 from Griffithsia sp. and functional homologsthereof, referred to collectively as “griffithsin.” Herein the term“griffithsin” is used generically to refer to a natural griffithsin orany related, functionally equivalent (i.e., anti-viral) polypeptide orderivative thereof. By definition, in this context, a related,functionally equivalent polypeptide or derivative thereof (a) contains asequence of at least eight contiguous amino acids directly identical toa sub-sequence of eight contiguous amino acids contained within anatural griffithsin, and (b) can specifically bind to a virus, inparticular an influenza virus or a retrovirus, more specifically aprimate immunodeficiency virus, more specifically HIV-1, HIV-2 or SIV,or to an infected host cell expressing one or more viral antigen(s),more specifically an envelope glycoprotein, such as gp120, of therespective virus. In addition, such a functionally equivalentpolypeptide or derivative thereof can comprise the amino acid sequenceof a natural griffithsin (see SEQ ID NO:3), in which 1-20, preferably1-10, more preferably 1, 2, 3, 4, or 5, and most preferably 1 or 2,amino acids have been removed from one or both ends, preferably fromonly one end, e.g., removed from the amino-terminal end, of naturalgriffithsin. Alternatively, a functionally equivalent polypeptide orderivative thereof can comprise the amino acid sequence of a nativegriffithsin (see SEQ ID NO:3), in which 1-20, preferably 1-10, morepreferably 1, 2, 3, 4, or 5, and most preferably 1 or 2, amino acidshave been added to one or both ends, preferably from only one end, e.g.,the amino-terminal end, of the native griffithsin.

The invention further provides an isolated and purified polypeptideencoded by a nucleic acid molecule comprising a sequence of SEQ ID NO:1or a nucleic acid molecule encoding an amino acid sequence of SEQ IDNO:2 or SEQ ID NO:3. Upon examination of the antiviral griffithsinpolypeptide, the amino acid at position 31 of SEQ ID NO:3 (representedas Xaa) was found not to be a familiar amino acid residue. Placement ofan alanine at position 31, such as achieved in the recombinantgriffithsin polypeptide described herein (SEQ ID NO:2), results in apolypeptide exhibiting equivalent activity as the natural griffithsinpolypeptide. If desired, the amino acid at position 31 can besubstituted with any other amino acid to facilitate protein production.Ideally, the substitution at position 31 of SEQ ID NO:3 does notdiminish the anti-viral activity of the protein (e.g., does not diminishthe anti-viral activity more than 50%, more than 30% or more than 10%)as compared to the anti-viral activity of the native protein.Preferably, the aforementioned nucleic acid molecules encode at leasteight (e.g., at least 10, at least 20, at least 30, at least 50, atleast 70, at least 80, at least 90, or at least 100) contiguous aminoacids of the amino acid sequence of SEQ ID NO:3, which desirably haveanti-viral activity. If the at least eight contiguous amino acids of SEQID NO:3 comprise amino acids 1-121, desirably amino acid residue 45, 60,71, and/or 104 has been rendered glycosylation resistant, whilemaintaining antiviral activity of the polypeptide.

The term “isolated” as used herein means having been removed from itsnatural environment. The term “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term and not tobe construed as absolute purity. By “antiviral” is meant that thepolypeptide or fragment thereof can inhibit a virus (e.g., inhibit entryof a virus into a host cell, limit the spread of viral infection byinhibiting cell to cell fusion, and the like), in particular aninfluenza virus, such as influenza virus or a strain A or strain B, or aretrovirus, specifically a primate immunodeficiency virus, morespecifically a human immunodeficiency virus (HIV), such as HIV-1, HIV-2or SIV.

Preferably, the polypeptide or derivative thereof comprises an aminoacid sequence that is substantially homologous to that of an anti-viralprotein from Griffithsia sp. By “substantially homologous” is meantsufficient homology to render the polypeptide or derivative thereofanti-viral, with anti-viral activity characteristic of an anti-viralprotein isolated from Griffithsia sp. At least about 50% homology (e.g.,at least about 60% homology, at least about 65% homology, or at leastabout 70% homology), preferably at least about 75% homology (e.g., atleast about 80% homology or at least about 85% homology), and mostpreferably at least about 90% homology (e.g., at least about 95%homology) should exist.

Alterations of the natural amino acid sequence to produce variantpolypeptides can be done by a variety of means known to those skilled inthe art. For instance, amino acid substitutions can be convenientlyintroduced into the polypeptides at the time of synthesis.Alternatively, site-specific mutations can be introduced by ligatinginto an expression vector a synthesized oligonucleotide comprising themodified site. Alternately, oligonucleotide-directed, site-specificmutagenesis procedures can be used, such as disclosed in Walder et al.,Gene, 42: 133 (1986); Bauer et al., Gene, 37: 73 (1985); Craik,Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and4,737,462.

It is within the skill of the ordinary artisan to select synthetic andnaturally-occurring amino acids that effect conservative or neutralsubstitutions for any particular naturally-occurring amino acids. Theordinarily skilled artisan desirably will consider the context in whichany particular amino acid substitution is made, in addition toconsidering the hydrophobicity or polarity of the side-chain, thegeneral size of the side chain and the pK value of side-chains withacidic or basic character under physiological conditions. For example,lysine, arginine, and histidine are often suitably substituted for eachother, and more often arginine and histidine. As is known in the art,this is because all three amino acids have basic side chains, whereasthe pK value for the side-chains of lysine and arginine are much closerto each other (about 10 and 12) than to histidine (about 6). Similarly,glycine, alanine, valine, leucine, and isoleucine are often suitablysubstituted for each other, with the proviso that glycine is frequentlynot suitably substituted for the other members of the group. This isbecause each of these amino acids are relatively hydrophobic whenincorporated into a polypeptide, but glycine's lack of an α-carbonallows the phi and psi angles of rotation (around the α-carbon) so muchconformational freedom that glycinyl residues can trigger changes inconformation or secondary structure that do not often occur when theother amino acids are substituted for each other. Other groups of aminoacids frequently suitably substituted for each other include, but arenot limited to, the group consisting of glutamic and aspartic acids; thegroup consisting of phenylalanine, tyrosine and tryptophan; and thegroup consisting of serine, threonine and, optionally, tyrosine.Additionally, the ordinarily skilled artisan can readily group syntheticamino acids with naturally-occurring amino acids.

The ordinarily skilled artisan can generate griffithsin mutants orvariants by, for example, substituting or mutating amino acids which arenot critical for the anti-viral function of the polypeptide. Ideally,mutations that do not modify the electronic or structural environment ofthe peptide are generated to retain optimal antiviral activity. Forexample, natural griffithsin forms dimers, which can be advantageous insome embodiments. Therefore, alterations which do not disrupt dimerformation can be preferred. Amino acid residues which are notresponsible for folding or stability of the three-dimensionalconformation of the griffithsin polypeptide are candidate residues formutation. Alternatively or in addition, amino acids which are notinvolved in glycoprotein binding can be mutated or replaced. It isunderstood that surface hydrophobicity plays a key role inprotein-protein interactions and surface electrophilicity is importantto protein-sugar interactions, such as the interaction betweengriffithsin and viral proteins. Hydrophobic surface clusters andelectrophilic surface clusters on the griffithsin peptide or homologswhich suggest regions critical for interaction with the viral envelopecan be mapped using routine methods such as those disclosed in Bewley etal., Nature Structural Biology, 5(7): 571-578 (1998). Amino acidresidues not found either in electrophilic or hydrophobic surfaceclusters are likely not critical for hydrophobicity or electrophilicityof these clusters and, thus, are appropriate targets for mutation tocreate griffithsin fragments (e.g., anti-viral polypeptides comprisingat least about eight contiguous amino acids of SEQ ID NO:2 or SEQ IDNO:3), variants, mutants, or homologs (e.g., griffithsin variants having80%, 85%, or 90% homology to SEQ ID NO:2 or SEQ ID NO:3) which retainantiviral activity. If desired, amino acid residues which areresponsible for binding to high-mannose oligosaccharide-containingglycoproteins on the viral surface can be mutated to increase thespecificity or affinity of glycoprotein binding.

If desired, the proteins and peptides of the invention (includingantiviral fragments, variant polypeptides, fusion proteins, andconjugates) can be modified, for instance, by glycosylation, amidation,carboxylation, or phosphorylation, or by the creation of acid additionsalts, amides, esters, in particular C-terminal esters, and N-acylderivatives of the proteins of the invention. The polypeptides also canbe modified to create protein derivatives by forming covalent ornoncovalent complexes with other moieties in accordance with methodsknown in the art. Covalently-bound complexes can be prepared by linkingthe chemical moieties to functional groups on the side chains of aminoacids comprising the proteins, or at the N- or C-terminus. Desirably,such modifications and conjugations do not adversely affect the activityof the polypeptides (and variants thereof). While such modifications andconjugations can have greater or lesser activity, the activity desirablyis not negated and is characteristic of the unaltered polypeptide.

The polypeptides (and fragments, homologs, variants, and fusionproteins) can be prepared by any of a number of conventional techniques.The polypeptide can be isolated or purified from a naturally occurringsource or from a recombinant source. For instance, in the case ofrecombinant proteins, a DNA fragment encoding a desired polypeptide canbe subcloned into an appropriate vector using well-known moleculargenetic techniques (see, e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory (1989)) andother references cited herein under “EXAMPLES”). The fragment can betranscribed and the polypeptide subsequently translated in vitro.Commercially available kits also can be employed (e.g., such asmanufactured by Clontech, Palo Alto, Calif.; Amersham Life Sciences,Inc., Arlington Heights, Ill.; InVitrogen, San Diego, Calif.; and thelike). The polymerase chain reaction optionally can be employed in themanipulation of nucleic acids.

Such polypeptides also can be synthesized using an automated peptidesynthesizer in accordance with methods known in the art. Alternately,the polypeptide (and fragments, homologs, variants, and fusion proteins)can be synthesized using standard peptide synthesizing techniqueswell-known to those of skill in the art (e.g., as summarized inBodanszky, Principles of Peptide Synthesis, (Springer-Verlag,Heidelberg:1984)). In particular, the polypeptide can be synthesizedusing the procedure of solid-phase synthesis (see, e.g., Merrifield, J.Am. Chem. Soc., 85: 2149-54 (1963); Barany et al., Int. J. PeptideProtein Res., 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). Ifdesired, this can be done using an automated peptide synthesizer.Removal of the t-butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups andseparation of the polypeptide from the resin can be accomplished by, forexample, acid treatment at reduced temperature. The protein-containingmixture then can be extracted, for instance, with diethyl ether, toremove non-peptidic organic compounds, and the synthesized polypeptidecan be extracted from the resin powder (e.g., with about 25% w/v aceticacid). Following the synthesis of the polypeptide, further purification(e.g., using HPLC) optionally can be preformed in order to eliminate anyincomplete proteins, polypeptides, peptides or free amino acids. Aminoacid and/or HPLC analysis can be performed on the synthesizedpolypeptide to validate its identity. For other applications accordingto the invention, it may be preferable to produce the polypeptide aspart of a larger fusion protein, either by chemical conjugation orthrough genetic means, such as are known to those skilled in the art. Inthis regard, the invention also provides a fusion protein comprising theisolated or purified antiviral polypeptide (or fragment thereof) orvariant thereof and one or more other protein(s) having any desiredproperties or effector functions, such as cytotoxic or immunologicalproperties, or other desired properties, such as to facilitateisolation, purification, analysis, or stability of the fusion protein.

A griffithsin conjugate comprising a griffithsin coupled to at least oneeffector component, which can be the same or different, is alsoprovided. The effector component can be polyethylene glycol, dextran,albumin, an immunological reagent, a toxin, an antiviral agent, or asolid support matrix. “Immunological reagent” will be used to refer toan antibody, an antibody fragment (e.g., an F(ab′)₂, an Fab′, an Fab, anFv, an sFv, a dsFv, or an Fc antibody fragment), an immunoglobulin, andan immunological recognition element. An immunological recognitionelement is an element, such as a peptide, e.g., the FLAG sequence of arecombinant griffithsin-FLAG fusion protein, which facilitates, throughimmunological recognition, isolation and/or purification and/or analysisof the protein or peptide to which it is attached. An immunologicalreagent also can be an immunogenic peptide, which can be fused togriffithsin for enhancing an immune response. In this respect, theinvention provides an anti-viral conjugate comprising a griffithsinpolypeptide or fragment thereof bound to a virus or viral envelopeglycoprotein. A griffithsin fusion protein is a type of griffithsinconjugate, wherein a griffithsin is coupled to one or more otherprotein(s) having any desired properties or effector functions, such ascytotoxic or immunological properties, or other desired properties, suchas to facilitate isolation, purification or analysis of the fusionprotein or increase the stability or in vivo half-life of the fusionprotein. Griffithsin also can be attached to a chemical moiety whichallows recognition, isolation, purification, and/or analysis of theprotein or peptide. An example of such a chemical moiety is a His tag ofa recombinant griffithsin-His fusion protein.

A “toxin” can be, for example, Pseudomonas exotoxin. An “antiviralagent” can be AZT, ddI, ddC, 3TC gancyclovir, fluorinateddideoxynucleosides, nevirapine, R82913, Ro 31-8959, BI-RJ-70, acyclovir,α-interferon, recombinant sCD4, michellamines, calanolides, nonoxynol-9,gossypol and derivatives thereof, gramicidin, amantatadine, rimantadine,and neuraminidase inhibitors, and cyanovirin-N or a functional homologor derivative thereof (see, for example, U.S. Pat. No. 5,843,882). A“solid support matrix” can be a magnetic bead, a flow-through matrix, asponge, a stent, a culture plate, or a matrix comprising a contraceptivedevice, such as a condom, diaphragm, cervical cap, vaginal ring orcontraceptive sponge. In an alternative embodiment, a solid supportmatrix can be an implant for surgical implantation in a host and, ifappropriate, later removal.

In view of the foregoing, the invention further provides a compositioncomprising (i) the isolated or purified antiviral polypeptide (orfragment thereof), a variant thereof, a fusion protein of the antiviralpolypeptide (or fragment thereof) or variant thereof, and a conjugate ofthe antiviral polypeptide (or fragment thereof) or variant thereof,and/or (ii) a carrier, excipient or adjuvant therefor. Preferably,component (i) of the composition is present in an antiviral effectiveamount and the carrier is pharmaceutically acceptable. By “antiviraleffective amount” is meant an amount sufficient to inhibit theinfectivity of the virus.

The carrier can be any of those conventionally used and is limited onlyby chemico-physical considerations, such as solubility and lack ofreactivity with the active agent of the invention, and by the route ofadministration. It is preferred that the pharmaceutically acceptablecarrier be one which is chemically inert to the active agent and onewhich has no detrimental side effects or toxicity under the conditionsof use. The pharmaceutically acceptable carriers described herein, forexample, vehicles, adjuvants, excipients, and diluents, are well-knownto those ordinarily skilled in the art and are readily available to thepublic. Typically, the composition, such as a pharmaceuticalcomposition, can comprise a physiological saline solution; dextrose orother saccharide solution; or ethylene, propylene, polyethylene, orother glycol. The pharmaceutical composition preferably does notcomprise mannose or N-acetyl-glucosamine, as these molecules mayinterfere with the functioning of the antiviral agent.

The invention also provides a method of obtaining a griffithsin fromGriffithsia sp. Such a method comprises (a) identifying an extract ofGriffithsia sp. containing anti-viral activity, (b) optionally removinghigh molecular weight biopolymers from the extract, (c) anti-viralbioassay-guided fractionating the extract to obtain a crude extract ofgriffithsin, and (d) purifying the crude extract by reverse-phase HPLCto obtain griffithsin (see, also, Example 1). More specifically, themethod involves the use of ethanol to remove high molecular weightbiopolymers from the extract and the use of an anti-HIV bioassay toguide fractionation of the extract.

Griffithsin (a polypeptide of exactly SEQ ID NO:3), which was isolatedand purified using the aforementioned method, was subjected toconventional procedures typically used to determine the amino acidsequence of a given pure protein. Thus, the griffithsin was initiallysequenced by N-terminal Edman degradation of intact protein and numerousoverlapping peptide fragments generated by endoproteinase digestion.Amino acid analysis was in agreement with the deduced sequence. ESI massspectrometry of reduced, HPLC-purified griffithsin showed a molecularion consistent with the calculated value. These studies indicated thatgriffithsin from Griffithsia was comprised of a unique sequence of 121amino acids having little or no significant homology or identity topreviously described proteins or transcription products of knownnucleotide sequences. No more than eight contiguous amino acids fromgriffithsin were found in any amino acid sequences from known proteins,nor were there any known proteins from any source having significantsequence identity with griffithsin. Given the chemically deduced aminoacid sequence of griffithsin, a corresponding recombinant griffithsin(r-griffithsin) was created and used to establish definitively that thededuced amino acid sequence was, indeed, active against virus, such asHIV and influenza.

Accordingly, the invention provides isolated and purified nucleic acidmolecules and synthetic nucleic acid molecules, which comprise a codingsequence for a griffithsin, such as an isolated and purified nucleicacid molecule comprising a sequence of SEQ ID NO:1, an isolated andpurified nucleic acid molecule encoding an amino acid sequence of SEQ IDNO:2, an isolated and purified nucleic acid sequence encoding an aminoacid sequence SEQ ID NO:3, an isolated and purified nucleic acidmolecule comprising a sequence of SEQ ID NO:4, an isolated and purifiednucleic acid sequence encoding an amino acid sequence of SEQ ID NO:5,and a nucleic acid molecule that is substantially homologous orsubstantially identical to any one of the aforementioned nucleic acidmolecules. By “substantially homologous” is meant sufficient homology torender the polypeptide or derivative thereof anti-viral, with anti-viralactivity characteristic of an anti-viral protein isolated fromGriffithsia. At least about 50% homology or identity (e.g., at leastabout 60%, at least about 65%, or at least about 70% homology oridentity), preferably at least about 75% homology or identity (e.g., atleast about 80% or at least about 85% homology or identity), and mostpreferably at least about 90% homology or identity (e.g., at least about95% homology or identity) should exist.

The inventive nucleic acid molecule preferably comprises a nucleic acidsequence encoding at least eight (preferably at least 10, morepreferably at least 20, and most preferably at least 30) contiguousamino acids of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:2.The inventive nucleic acid molecule also comprises a nucleic acidsequence encoding a polypeptide comprising the amino acid sequence of anative griffithsin, in which 1-20, preferably 1-10, more preferably 1,2, 3, 4, or 5, and most preferably 1 or 2, amino acids have been removedfrom one or both ends, preferably from only one end, e.g., removed fromthe amino-terminal end, of the native griffithsin. Alternatively, thenucleic acid molecule can comprise a nucleic acid sequence encoding apolypeptide comprising the amino acid sequence of a natural griffithsin(see SEQ ID NO:3), in which 1-20, preferably 1-10, more preferably 1, 2,3, 4, or 5, and most preferably 1 or 2, amino acids have been added toone or both ends, preferably from only one end, e.g., the amino-terminalend, of the native griffithsin. Preferably, the isolated and purifiednucleic acid molecule encodes a polypeptide comprising at least eightcontiguous amino acids of SEQ ID NO:3, which desirably have anti-viralactivity. If the at least eight contiguous amino acids comprise aminoacids 1-121 of SEQ ID NO:3, desirably amino acids 46, 60, 71, and/or 104have been rendered glycosylation resistant, while maintaining antiviralactivity of the polypeptide. Deletions and substitutions of SEQ ID NO:2or SEQ ID NO:3 are within the skill in the art.

Given the present disclosure, it will be apparent to one skilled in theart that a partial griffithsin gene sequence will likely suffice to codefor a fully functional, i.e., anti-viral, such as anti-influenza oranti-HIV, griffithsin. A minimum essential DNA coding sequence(s) for afunctional griffithsin can readily be determined by one skilled in theart, for example, by synthesis and evaluation of sub-sequencescomprising the native griffithsin, and by site-directed mutagenesisstudies of the griffithsin DNA coding sequence.

Using an appropriate DNA coding sequence, a recombinant griffithsin canbe made by genetic engineering techniques (for general background see,e.g., Nicholl, in An Introduction to Genetic Engineering, CambridgeUniversity Press: Cambridge (1994), pp. 1-5 & 127-130; Steinberg et al.,in Recombinant DNA Technology Concepts and Biomedical Applications,Prentice Hall: Englewood Cliffs, N.J. (1993), pp. 81-124 & 150-162;Sofer in Introduction to Genetic Engineering, Butterworth-Heinemann,Stoneham, Mass. (1991), pp. 1-21 & 103-126; Old et al., in Principles ofGene Manipulation, Blackwell Scientific Publishers: London (1992), pp.1-13 & 108-221; and Emtage, in Delivery Systems for Peptide Drugs, Daviset al., eds., Plenum Press: New York (1986), pp. 23-33). For example, aGriffithsia gene or cDNA encoding a griffithsin can be identified andsubcloned. The gene or cDNA then can be incorporated into an appropriateexpression vector and delivered into an appropriatepolypeptide-synthesizing organism (e.g., E. coli, S. cerevisiae, P.pastoris, or other bacterial, yeast, insect, plant or mammalian cells),where the gene, under the control of an endogenous or exogenouspromoter, can be appropriately transcribed and translated.Alternatively, the expression vector can be administered to a plant oranimal, for example, for large-scale production (see, e.g., Fischer etal., Transgenic Res., 9 (4-5):279-299 (2000); Fischer et al., J. Biol.Regul. Homeost. Agents, 14: 83-92 (2000); deWilde et al., Plant Molec.Biol., 43: 347-359 (2000); Houdebine, Transgenic Research, 9: 305-320(2000); Brink et al., Theriogenology, 53: 139-148 (2000); Pollock etal., J. Immunol. Methods, 231: 147-157 (1999); Conrad et al., PlantMolec. Biol., 38: 101-109 (1998); Staub et al., Nature Biotech., 18:333-338 (2000); McCormick et al., PNAS USA, 96: 703-708 (1999); Zeitlinet al., Nature Biotech., 16: 1361-1364 (1998); Tacker et al., Microbesand Infection, 1: 777-783 (1999); Tacket et al., Nature Med.,4(5):607-609 (1998); and Methods in Biotechnology, Recombinant Proteinsfrom Plants, Production and Isolation of Clinically Useful Compounds,Cunningham and Porter, eds., Humana Press: Totowa, N.J. (1998)). Suchexpression vectors (including, but not limited to, phage, cosmid, viral,and plasmid vectors) are known to those skilled in the art, as arereagents and techniques appropriate for gene transfer (e.g.,transfection, electroporation, transduction, micro-injection,transformation, etc.). If a griffithsin is to be recombinantly producedin isolated eukaryotic cells or in a eukaryotic organism, such as aplant (see above references and also Methods in Biotechnology,Recombinant Proteins from Plants, Production and Isolation of ClinicallyUseful Compounds, Cunningham and Porter, eds., Humana Press: Totowa,N.J. (1998)), desirably the N-linked glycosylation sites at positions45, 60, 71, and/or 104 is rendered glycosylation-resistant, such as inaccordance with the methods described herein. Subsequently, therecombinantly produced polypeptide can be isolated and purified usingstandard techniques known in the art (e.g., chromatography,centrifugation, differential solubility, isoelectric focusing, etc.),and assayed for anti-viral activity.

Alternatively, a natural griffithsin can be obtained from Griffithsia bynon-recombinant methods, and sequenced by conventional techniques. Thesequence can then be used to synthesize the corresponding DNA, which canbe subcloned into an appropriate expression vector and delivered into apolypeptide-producing cell for en mass recombinant production of thedesired polypeptide.

In this regard, the invention also provides a vector comprising a DNAsequence, e.g., a Griffithsia gene sequence for griffithsin, a cDNAencoding a griffithsin, or a synthetic DNA sequence encodinggriffithsin. The vector can be targeted to a cell-surface receptor if sodesired. A nucleic acid molecule as described above can be cloned intoany suitable vector and can be used to transform or transfect anysuitable host. The selection of vectors and methods to construct themare commonly known to persons of ordinary skill in the art and aredescribed in general technical references (see, in general, “RecombinantDNA Part D,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds.,Academic Press (1987) and the references cited herein under “EXAMPLES”).Desirably, the vector comprises regulatory sequences, such astranscription and translation initiation and termination codons, whichare specific to the type of host (e.g., bacterium, fungus, plant oranimal) into which the vector is to be introduced, as appropriate andtaking into consideration whether the vector is DNA or RNA. Preferably,the vector comprises regulatory sequences that are specific to the genusof the host. Most preferably, the vector comprises regulatory sequencesthat are specific to the species of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid as described above or a portion thereofligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from Co1E1, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

One of ordinary skill in the art will appreciate that any of a number ofvectors known in the art are suitable for use in the invention. Suitablevectors include those designed for propagation and expansion or forexpression or both. Examples of suitable vectors include, for instance,plasmids, plasmid-liposome complexes, and viral vectors, e.g.,parvoviral-based vectors (i.e., adeno-associated virus (AAV)-basedvectors), retroviral vectors, herpes simplex virus (HSV)-based vectors,and adenovirus-based vectors. Any of these expression constructs can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994);Fischer et al., Transgenic Res., 9 (4-5):279-299 (2000); Fischer et al.,J. Biol. Regul. Homeost. Agents, 14: 83-92 (2000); deWilde et al., PlantMolec. Biol., 43: 347-359 (2000); Houdebine, Transgenic Research, 9:305-320 (2000); Brink et al., Theriogenology, 53: 139-148 (2000);Pollock et al., J. Immunol. Methods, 231: 147-157 (1999); Conrad et al.,Plant Molec. Biol., 38: 101-109 (1998); Staub et al., Nature Biotech.,18: 333-338 (2000); McCormick et al., PNAS USA, 96: 703-708 (1999);Zeitlin et al., Nature Biotech., 16: 1361-1364 (1998); Tacker et al.,Microbes and Infection, 1: 777-783 (1999); and Tacket et al., NatureMed., 4(5):607-609 (1998). Examples of cloning vectors include the pUCseries, the pBluescript series (Stratagene, LaJolla, Calif.), the pETseries (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, Calif.).Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λ NM1149, also can be used. Examples of plant expressionvectors include pBI101, pBI101.2, pBI101.3, pBI121 and pBIN19(Clonetech, Palo Alto, Calif.). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clonetech).

An expression vector can comprise a native or normative promoteroperably linked to an isolated or purified nucleic acid as describedabove. The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the skill in theart. Similarly, the combining of a nucleic acid molecule as describedabove with a promoter is also within the skill in the art.

The DNA, whether isolated and purified or synthetic, or cDNA encoding agriffithsin can encode for either the entire griffithsin or a portionthereof. Where the DNA or cDNA does not comprise the entire codingsequence of the native griffithsin, the DNA or cDNA can be subcloned aspart of a gene fusion. In a transcriptional gene fusion, the DNA or cDNAwill contain its own control sequence directing appropriate productionof protein (e.g., ribosome binding site, translation initiation codon,etc.), and the transcriptional control sequences (e.g., promoterelements and/or enhancers) will be provided by the vector. In atranslational gene fusion, transcriptional control sequences as well asat least some of the translational control sequences (i.e., thetranslational initiation codon) will be provided by the vector. In thecase of a translational gene fusion, a chimeric protein will beproduced.

Genes also can be constructed for specific fusion proteins containing afunctional griffithsin component plus a fusion component conferringadditional desired attribute(s) to the composite protein. For example, afusion sequence for a toxin or immunological reagent can be added tofacilitate purification and analysis of the functional protein.

Genes can be specifically constructed to code for fusion proteins, whichcontain a griffithsin coupled to an effector protein, such as a toxin orimmunological reagent, for specific targeting to a virus orviral-infected cells, e.g., HIV and/or HIV-infected cells or influenzaand/or influenza-infected cells. In these instances, the griffithsinmoiety serves not only as a neutralizing agent but also as a targetingagent to direct the effector activities of these molecules selectivelyagainst a given virus, such as HIV or influenza. Thus, for example, atherapeutic agent can be obtained by combining the HIV-targetingfunction or influenza-targeting function of a functional griffithsinwith a toxin aimed at neutralizing infectious virus and/or by destroyingcells producing infectious virus, such as HIV or influenza. Similarly, atherapeutic agent can be obtained, which combines the viral-targetingfunction of a griffithsin with the multivalency and effector functionsof various immunoglobulin subclasses. Example 6 further illustrates theviral-targeting, specifically gp120-targeting, properties of agriffithsin.

Similar rationales underlie extensive developmental therapeutic effortsexploiting the HIV gp120-targeting properties of sCD4. For example,sCD4-toxin conjugates have been prepared in which sCD4 is coupled to aPseudomonas exotoxin component (Chaudhary et al., in The HumanRetrovirus, Gallo et al., eds., Academic Press: San Diego, Calif.(1991), pp. 379-387; and Chaudhary et al., Nature, 335: 369-372 (1988)),or to a diphtheria toxin component (Aullo et al., EMBO J., 11: 575-583(1992)) or to a ricin A-chain component (Till et al., Science, 242:1166-1167 (1988)). Likewise, sCD4-immunoglobulin conjugates have beenprepared in attempts to decrease the rate of in vivo clearance offunctional sCD4 activity, to enhance placental transfer, and to effect atargeted recruitment of immunological mechanisms of pathogenelimination, such as phagocytic engulfment and killing byantibody-dependent cell-mediated cytotoxicity, to kill and/or removeHIV-infected cells and virus (Capon et al., Nature, 337: 525-531 (1989);Traunecker et al., Nature, 339: 68-70 (1989); and Langner et al. (1993),supra). While such CD4-immunoglobulin conjugates (sometimes called“immunoadhesins”) have, indeed, shown advantageous pharmacokinetic anddistributional attributes in vivo, and anti-HIV effects in vitro,clinical results have been discouraging (Schooley et al. (1990), supra;Husson et al. (1992), supra; and Langner et al. (1993), supra). This isnot surprising since clinical isolates of HIV, as opposed to laboratorystrains, are highly resistant to binding and neutralization by sCD4(Orloff et al. (1995), supra; and Moore et al. (1992), supra). Thegriffithsin polypeptide binds to a wide range of sugars present on viralglycoproteins and, therefore, can inhibit a wide range of viruses whichdisplay those glycoproteins. The extraordinarily broad targetingproperties of a functional griffithsin to viruses, e.g., primateretroviruses, in general, and clinical and laboratory strains, inparticular, can be especially advantageous for combining with toxins,immunoglobulins and other selected effector proteins.

Viral-targeted conjugates can be prepared either by genetic engineeringtechniques (see, for example, Chaudhary et al. (1988), supra) or bychemical coupling of the targeting component with an effector component.The most feasible or appropriate technique to be used to construct agiven griffithsin conjugate or fusion protein will be selected basedupon consideration of the characteristics of the particular effectormolecule selected for coupling to a griffithsin. For example, with aselected non-proteinaceous effector molecule, chemical coupling, ratherthan genetic engineering techniques, may be the only feasible option forcreating the desired griffithsin conjugate.

Accordingly, the invention also provides nucleic acid molecules encodinggriffithsin fusion proteins. In particular, the invention provides anucleic acid molecule comprising SEQ ID NO:4 and substantiallyhomologous sequences thereof. Also provided is a vector comprising anucleic acid sequence encoding a griffithsin fusion protein and a methodof obtaining a griffithsin fusion protein by expression of the vectorencoding a griffithsin fusion protein in a protein-synthesizing organismas described above. Accordingly, griffithsin fusion proteins are alsoprovided.

In view of the above, the invention further provides an isolated andpurified nucleic acid molecule, which comprises a griffithsin codingsequence, such as one of the aforementioned nucleic acids, namely anucleic acid molecule encoding an amino acid sequence of SEQ ID NO:2 orSEQ ID NO:3 or a nucleic acid molecule comprising a sequence of SEQ IDNO:1 coupled to a second nucleic acid encoding an effector protein. Thefirst nucleic acid preferably comprises a nucleic acid sequence encodingat least eight contiguous amino acids of the amino acid sequence of SEQID NO:2 or SEQ ID NO:3, which encodes a functional griffithsin, and thesecond nucleic acid preferably encodes an effector protein, such as atoxin or immunological reagent as described herein.

Accordingly, the invention also further provides an isolated andpurified fusion protein encoded by a nucleic acid molecule comprising asequence of SEQ ID NO:1 or a nucleic acid molecule encoding an aminoacid sequence of SEQ ID NO:2 or SEQ ID NO: 3, either one of which iscoupled to a second nucleic acid encoding an effector protein.Preferably, the aforementioned nucleic acid molecules encode at leasteight contiguous amino acids of the amino acid sequence of SEQ ID NO:2or SEQ ID NO:3, which desirably have anti-viral activity, coupled to aneffector molecule, such as a toxin or immunological reagent as describedabove. Preferably, the effector molecule targets a virus, morepreferably HIV or influenza, and, most preferably glycoprotein gp120 ofHIV or hemagluttinin of influenza. If the at least eight contiguousamino acids of SEQ ID NO:3 (or SEQ ID NO:2) comprise amino acids 1-121,desirably amino acids 46, 60, 71, and/or 104 have been renderedglycosylation-resistant, yet maintain antiviral activity by substitutionof the asparagine at those positions with, for example, an alanine or aglutamine residue.

The coupling can be effected at the DNA level or by chemical coupling asdescribed above. For example, a griffithsin-effector protein conjugateof the invention can be obtained by (a) selecting a desired effectorprotein or peptide; (b) synthesizing a composite DNA coding sequencecomprising a first DNA coding sequence comprising one of theaforementioned nucleic acid sequences, which codes for a functionalgriffithsin, coupled to a second DNA coding sequence for an effectorprotein or peptide, e.g., a toxin or immunological reagent; (c)expressing said composite DNA coding sequence in an appropriateprotein-synthesizing organism; and (d) purifying the desired fusionprotein to substantially pure form. Alternatively, agriffithsin-effector molecule conjugate of the invention can be obtainedby (a) selecting a desired effector molecule and a griffithsin orgriffithsin fusion protein; (b) chemically coupling the griffithsin orgriffithsin fusion protein to the effector molecule; and (c) purifyingthe desired griffithsin-effector molecule conjugate to substantiallypure form.

Conjugates comprising a functional griffithsin (e.g., an anti-viralpolypeptide comprising at least eight contiguous amino acids of SEQ IDNO:3, such as SEQ ID NO:3, wherein the at least eight contiguous aminoacids bind to a virus, in particular an infectious virus, such asinfluenza virus or HIV, in which case the griffithsin binds to gp120 orhemagluttinin) coupled to an anti-griffithsin antibody, a virus, a viralglycoprotein, or at least one effector component, which can be the sameor different, such as a toxin, an immunological reagent, an antiviralagent, or other functional reagent, can be designed even morespecifically to exploit the unique viral targeting, e.g.,gp120-targeting properties, of griffithsins.

Other functional reagents that can be used as effector components in theinventive conjugates can include, for example, polyethylene glycol,dextran, albumin, a solid support matrix, and the like, whose intendedeffector functions may include one or more of the following: to improvestability of the conjugate; to increase the half-life of the conjugate;to increase resistance of the conjugate to proteolysis; to decrease theimmunogenicity of the conjugate; to provide a means to attach orimmobilize a functional griffithsin onto a solid support matrix (e.g.,see, for example, Harris, in Poly(Ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, Harris, ed., Plenum Press: NewYork (1992), pp. 1-14). Conjugates furthermore can comprise a functionalgriffithsin coupled to more than one effector molecule, each of which,optionally, can have different effector functions (e.g., such as a toxinmolecule (or an immunological reagent) and a polyethylene glycol (ordextran or albumin) molecule). Diverse applications and uses offunctional proteins and peptides, such as in the present instance afunctional griffithsin, attached to or immobilized on a solid supportmatrix, are exemplified more specifically for poly(ethylene glycol)conjugated proteins or peptides in a review by Holmberg et al. (InPoly(Ethylene Glycol) Chemistry: Biotechnical and BiomedicalApplications, Harris, ed., Plenum Press: New York (1992), pp. 303-324).Preferred examples of solid support matrices include magnetic beads, aflow-through matrix, and a matrix comprising a contraceptive device,such as a condom, a diaphragm, a cervical cap, a vaginal ring or asponge.

Example 4 reveals novel gp120-directed effects of griffithsins.Solid-phase ELISA experiments show that griffithsin is capable of globalconformational effects on gp120, as observed as a decrease ofimmunoreactivity at multiple, distinct, non-overlapping epitopes.

The range of anti-viral activity of griffithsin against diverseCD4⁺-tropic immunodeficiency virus strains in various target cells isremarkable; virtually all tested strains of HIV-1, HIV-2 and SIV weresimilarly sensitive to griffithsin; clinical isolates and laboratorystrains showed essentially equivalent sensitivity. Cocultivation ofchronically infected and uninfected CEM-SS cells with griffithsin didnot inhibit viral replication, but did cause a concentration-dependentinhibition of cell-to-cell fusion and virus transmission; similarresults from binding and fusion inhibition assays employingHeLa-CD4-LTR-β-galactosidase cells were consistent with griffithsininhibition of virus-cell and/or cell-cell binding.

The anti-viral, e.g., anti-HIV, activity of the griffithsins andconjugates thereof of the invention can be further demonstrated in aseries of interrelated in vitro anti-viral assays (Gulakowski et al., J.Virol. Methods, 33: 87-100 (1991)), which accurately predict foranti-viral activity in humans. These assays measure the ability ofcompounds to prevent the replication of HIV and/or the cytopathiceffects of HIV on human target cells. These measurements directlycorrelate with the pathogenesis of HIV-induced disease in vivo. Theresults of the analysis of the anti-viral activity of griffithsins orconjugates, as set forth in Examples 5-7 and 9, predict accurately theanti-viral activity of these products in vivo in humans and, therefore,establish the utility of the invention. Furthermore, since the inventionalso provides methods of ex vivo use of griffithsins and conjugates, theutility of griffithsins and conjugates thereof is even more certain.

The griffithsins and conjugates thereof of the invention can be shown toinhibit a virus, specifically a retrovirus, more specifically animmunodeficiency virus, such as the human immunodeficiency virus, i.e.,HIV-1 or HIV-2. The griffithsins and conjugates of the invention can beused to inhibit other retroviruses as well as other viruses (see, e.g.,Principles of Virology: Molecular Biology, Pathogenesis, and Control,Flint et al., eds., ASM Press: Washington, D.C. (2000), particularlyChapter 19). Examples of viruses that may be treated in accordance withthe invention include, but are not limited to, Type C and Type Dretroviruses, HTLV-1, HTLV-2, HIV, FIV, FLV, SIV, MLV, BLV, BIV, equineinfectious virus, anemia virus, avian sarcoma viruses, such as Roussarcoma virus (RSV), hepatitis type A, B, non-A and non-B viruses,arboviruses, varicella viruses, human herpes virus (e.g., HHV-6),measles, mumps, filovirus (e.g., Ebola, such as Ebola strains Sudan,Zaire, Cote d'Ivoire, and Reston) and rubella viruses. Griffithsins andconjugate thereof also can be used to inhibit influenza viral infection(see, e.g., Fields Virology, third edition, Fields et al., eds.,Lippincott-Raven Publishers: Philadelphia, Pa. (1996), particularlyChapter 45) prophylactically and therapeutically in accordance with themethods set forth herein.

Thus, the invention further provides a composition comprising (i) one ormore of an above-described purified or isolated nucleic acid or variantthereof, optionally as part of an encoded fusion protein, and (ii) acarrier, excipient or adjuvant. Preferably, (i) is present in anantiviral effective amount and the composition is pharmaceuticallyacceptable. The composition can further comprise at least one additionalactive agent, such as an antiviral agent other than a griffithsin (orantiviral fragment, fusion protein or conjugate thereof), in anantiviral effective amount. Suitable antiviral agents include AZT, ddA,ddI, ddC, 3TC gancyclovir, fluorinated dideoxynucleosides, acyclovir,α-interferon, nonnucleoside analog compounds, such as nevirapine (Shihet al., PNAS, 88: 9878-9882, (1991)), TIBO derivatives, such as R82913(White et al., Antiviral Res., 16: 257-266 (1991)), Ro31-8959, BI-RJ-70(Merigan, Am. J. Med., 90 (Suppl. 4A):8S-17S (1991)), michellamines(Boyd et al., J. Med. Chem., 37: 1740-1745 (1994)) and calanolides(Kashman et al., J. Med. Chem., 35: 2735-2743 (1992)), nonoxynol-9,gossypol and derivatives, gramicidin, Enfurtide (i.e., T20),cyanovirin-N and functional homologs thereof (Boyd et al. (1997),supra). Other exemplary antiviral compounds include protease inhibitors(see R. C. Ogden and C. W. Flexner, eds., Protease Inhibitors in AIDSTherapy, Marcel Dekker, N Y (2001)), such as saquinavir (see I. B.Duncan and S. Redshaw, in R. C. Ogden and C. W. Flexner, supra, pp.27-48), ritonavir (see D. J. Kempf, in R. C. Ogden and C. W. Flexner,supra, pp. 49-64), indinavir (see B. D. Dorsey and J. P. Vacca, in R. C.Ogden and C. W. Flexner, supra, pp. 65-84), nelfinavir (see S. H. Reich,in R. C. Ogden and C. W. Flexner, supra, pp. 85-100), amprenavir (see R.D. Tung, in R. C. Ogden and C. W. Flexner, supra, pp. 101-118), andanti-TAT agents. If the composition is to be used to induce an immuneresponse, it comprises an immune response-inducing amount of theinventive agent and can further comprise an immunoadjuvant, such aspolyphosphazene polyelectrolyte.

The pharmaceutical composition can contain other pharmaceuticals, suchas virucides, immunomodulators, immunostimulants, antibiotics andabsorption enhancers. Exemplary immunomodulators and immunostimulantsinclude various interleukins, sCD4, cytokines, antibody preparations,blood transfusions, and cell transfusions. Exemplary antibiotics includeantifungal agents, antibacterial agents, and anti-Pneumocystitis carniiagents. Exemplary absorption enhancers include bile salts and othersurfactants, saponins, cyclodextrins, and phospholipids (Davis (1992),supra).

An isolated cell comprising an above-described purified or isolatednucleic acid or variant thereof, optionally in the form of a vector,which is optionally targeted to a cell-surface receptor, is alsoprovided. Examples of host cells include, but are not limited to, ahuman cell, a human cell line, E. coli, B. subtilis, P. aerugenosa, S.cerevisiae, and N. crassa. E. coli, in particular E. coli TB-1, TG-2,DH5α, XL-Blue MRF' (Stratagene), SA2821 and Y1090. Preferably, the cellis a mammalian cell, bacterium, or yeast. A preferred bacterium islactobacillus or other commensal microorganism. The above-describednucleic acid or variant thereof, optionally in the form of a vector, canbe introduced into a host cell using such techniques as transfection,electroporation, transduction, micro-injection, transformation, and thelike.

Accordingly, the invention provides a method of inhibitingprophylactically or therapeutically a viral infection, in particular aninfluenza viral infection or HIV infection, of a host. The methodcomprises administering to the host an effective amount of an anti-viralpolypeptide or anti-viral polypeptide conjugate comprising at leasteight contiguous amino acids of SEQ ID NO:3, wherein the at least eightcontiguous amino acids are nonglycosylated and have anti-viral activity,whereupon the viral infection is inhibited. The anti-viral polypeptidecan be derived from a griffithsin obtained from Griffithsia orrecombinantly produced in accordance with the methods described above.Nonglycosylated anti-viral polypeptides can be produced in prokaryoticcells/organisms. Amino acids 45, 60, 71, and/or 104 in suchnonglycosylated antiviral polypeptides can be deleted or substituted,for example, with alanine or glutamine. Nonglycosylated antiviralpolypeptides also can be produced in eukaryotic cells/organisms byexpressing a portion of a griffithsin, such as that of SEQ ID NO:3, thatdoes not contain a glycosylation site or all or a portion of agriffithsin, such as that of SEQ ID NO:3, which contains a glycosylationsite that has been rendered glycosylation-resistant as described andexemplified herein. When the viral infection is an influenza viralinfection and the anti-viral polypeptide or anti-viral polypeptideconjugate is administered topically to the host, preferably theanti-viral protein or anti-viral peptide is administered to therespiratory system of the host, preferably as an aerosol ormicroparticulate powder.

The prophylactic and therapeutic treatment of many viral infections,including influenza virus infections, is complicated by appearance ofvirus forms resistant to currently employed medications, such asneurominidase inhibitors. The inventive method is particularly useful inthis context, as the inventive anti-viral polypeptide or anti-viralpolypeptide conjugate binds a wide range of glycoproteins present on theviral surface. Accordingly, the inventive anti-viral polypeptide orconjugate thereof can be administered to an animal, preferably a human,dog, cat, bird, cow, pig, horse, lamb, mouse, or rat, in combinationwith other anti-viral agents to guard against the propagation ofanti-viral-resistant strains of virus. In addition, it is thought thatduring adaptive mutation (e.g., resistance to neuraminidase inhibitors),the level of glycosylation found at the viral surface increases in someviruses, such as influenza. Thus, in that the inventive anti-viral agentbinds sugars of viral surface glycoproteins, the inventive methodprovides a valuable complimentary therapy to current anti-viralregimens.

Griffithsins and conjugates thereof collectively comprise polypeptidesand proteins, and, as such, are particularly susceptible to hydrolysisof amide bonds (e.g., catalyzed by peptidases) and disruption ofessential disulfide bonds or formation of inactivating or unwanteddisulfide linkages (Carone et al., J. Lab. Clin. Med., 100:1-14 (1982)).There are various ways to alter molecular structure, if necessary, toprovide enhanced stability to the griffithsin or conjugate thereof(Wunsch, Biopolymers, 22: 493-505 (1983); and Samanen, in PolymericMaterials in Medication, Gebelein et al., eds., Plenum Press: New York(1985) pp. 227-242), which may be essential for preparation and use ofpharmaceutical compositions containing griffithsins or conjugatesthereof for therapeutic or prophylactic applications against viruses,e.g., HIV. Possible options for useful chemical modifications of agriffithsin or conjugate include, but are not limited to, the following(adapted from Samanen, J. M. (1985) supra): (a) olefin substitution, (b)carbonyl reduction, (c) D-amino acid substitution, (d) N-methylsubstitution, (e) C-methyl substitution, (f) C—C′-methylene insertion,(g) dehydro amino acid insertion, (h) retro-inverso modification, (i)N-terminal to C-terminal cyclization, and (j) thiomethylenemodification. Griffithsins and conjugates thereof also can be modifiedby covalent attachment of carbohydrate and polyoxyethylene derivatives,which are expected to enhance stability and resistance to proteolysis(Abuchowski et al., in Enzymes as Drugs, Holcenberg et al., eds., JohnWiley: New York (1981), pp. 367-378).

Other important general considerations for design of delivery strategysystems and compositions, and for routes of administration, for proteinand peptide drugs, such as griffithsins and conjugates thereof(Eppstein, CRC Crit. Rev. Therapeutic Drug Carrier Systems, 5: 99-139(1988); Siddiqui et al., CRC Crit. Rev. Therapeutic Drug CarrierSystems, 3: 195-208 (1987); Banga et al., Int. J. Pharmaceutics, 48:15-50 (1988); Sanders, Eur. J. Drug Metab. Pharmacokinetics, 15: 95-102(1990); and Verhoef, Eur. J. Drug Metab. Pharmacokinetics, 15: 83-93(1990)), also apply. The appropriate delivery system for a givengriffithsin or conjugate thereof will depend upon its particular nature,the particular clinical application, and the site of drug action. Aswith any protein or peptide drug, oral delivery of a griffithsin or aconjugate thereof will likely present special problems, due primarily toinstability in the gastrointestinal tract and poor absorption andbioavailability of intact, bioactive drug therefrom. Therefore,especially in the case of oral delivery, but also possibly inconjunction with other routes of delivery, it will be necessary to usean absorption-enhancing agent in combination with a given griffithsin orconjugate thereof A wide variety of absorption-enhancing agents havebeen investigated and/or applied in combination with protein and peptidedrugs for oral delivery and for delivery by other routes (Verhoef(1990), supra; van Hoogdalem, Pharmac. Ther., 44: 407-443 (1989); andDavis, J. Pharm. Pharmacol, 44 (Suppl. 1):186-190 (1992)). Mostcommonly, typical enhancers fall into the general categories of (a)chelators, such as EDTA, salicylates, and N-acyl derivatives ofcollagen, (b) surfactants, such as lauryl sulfate andpolyoxyethylene-9-lauryl ether, (c) bile salts, such as glycholate andtaurocholate, and derivatives, such as taurodihydrofusidate, (d) fattyacids, such as oleic acid and capric acid, and their derivatives, suchas acylcarnitines, monoglycerides and diglycerides, (e) non-surfactants,such as unsaturated cyclic ureas, (f) saponins, (g) cyclodextrins, and(h) phospholipids.

Other approaches to enhancing oral delivery of protein and peptidedrugs, such as the griffithsins and conjugates thereof, can includeaforementioned chemical modifications to enhance stability togastrointestinal enzymes and/or increased lipophilicity. Alternatively,or in addition, the protein or peptide drug can be administered incombination with other drugs or substances, which directly inhibitproteases and/or other potential sources of enzymatic degradation ofproteins and peptides. Yet another alternative approach to prevent ordelay gastrointestinal absorption of protein or peptide drugs, such asgriffithsins or conjugates, is to incorporate them into a deliverysystem that is designed to protect the protein or peptide from contactwith the proteolytic enzymes in the intestinal lumen and to release theintact protein or peptide only upon reaching an area favorable for itsabsorption. A more specific example of this strategy is the use ofbiodegradable microcapsules or microspheres, both to protect vulnerabledrugs from degradation, as well as to effect a prolonged release ofactive drug (Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York (1984), pp. 1-60, 88-89,208-211). Microcapsules also can provide a useful way to effect aprolonged delivery of a protein and peptide drug, such as a griffithsinor conjugate thereof, after injection (Maulding, J. Controlled Release,6: 167-176 (1987)).

Given the aforementioned potential complexities of successful oraldelivery of a protein or peptide drug, it is fortunate that there arenumerous other potential routes of delivery of a protein or peptidedrug, such as a griffithsin or conjugate thereof. These routes includetopical, subcutaneous, intravenous, intraarterial, intrathecal,intracisternal, buccal, rectal, nasal, pulmonary, transdermal, vaginal,ocular, and the like (Eppstein (1988), supra; Siddiqui et al. (1987),supra; Banga et al. (1988), supra; Sanders (1990), supra; Verhoef(1990), supra; Barry, in Delivery Systems for Peptide Drugs, Davis etal., eds., Plenum Press: New York (1986), pp. 265-275; and Patton etal., Adv. Drug Delivery Rev, 8: 179-196 (1992)). With any of theseroutes, or, indeed, with any other route of administration orapplication, a protein or peptide drug, such as a griffithsin orconjugate thereof, may initiate an immunogenic reaction. In suchsituations it may be necessary to modify the molecule in order to maskimmunogenic groups. It also can be possible to protect against undesiredimmune responses by judicious choice of method of formulation and/oradministration. For example, site-specific delivery can be employed, aswell as masking of recognition sites from the immune system by use orattachment of a so-called tolerogen, such as polyethylene glycol,dextran, albumin, and the like (Abuchowski et al. (1981), supra;Abuchowski et al., J. Biol. Chem., 252: 3578-3581 (1977); Lisi et al.,J. Appl. Biochem, 4: 19-33 (1982); and Wileman et al., J. Pharm.Pharmacol, 38: 264-271 (1986)). Such modifications also can haveadvantageous effects on stability and half-life both in vivo and exvivo.

Procedures for covalent attachment of molecules, such as polyethyleneglycol, dextran, albumin and the like, to proteins, such as griffithsinsor conjugates thereof, are well-known to those skilled in the art, andare extensively documented in the literature (e.g., see Davis et al., inPeptide and Protein Drug Delivery, Lee, ed., Marcel Dekker: New York(1991), pp. 831-864).

Other strategies to avoid untoward immune reactions also can include theinduction of tolerance by administration initially of only low doses. Inany event, it will be apparent from the present disclosure to oneskilled in the art that for any particular desired medical applicationor use of a griffithsin or conjugate thereof, the skilled artisan canselect from any of a wide variety of possible compositions, routes ofadministration, or sites of application, what is advantageous.

Accordingly, the anti-viral griffithsins and conjugates thereof of theinvention can be formulated into various compositions for use, forexample, either in therapeutic treatment methods for infectedindividuals, or in prophylactic methods against viral, e.g., HIV andinfluenza virus, infection of uninfected individuals.

The invention also provides a composition, such as a pharmaceuticalcomposition, which comprises an isolated and purified griffithsin, agriffithsin conjugate, a matrix-anchored griffithsin or amatrix-anchored griffithsin conjugate, such as an anti-viral effectiveamount thereof. The composition can further comprise a carrier, such asa pharmaceutically acceptable carrier. The composition can furthercomprise at least one additional anti-viral compound other than agriffithsin or conjugate thereof, such as in an anti-viral effectiveamount of an anti-viral compound. Suitable anti-viral compounds includecyanovirin, AZT, ddI, ddC, gancyclovir, fluorinated dideoxynucleosides,nevirapine, R82913, Ro 31-8959, BI-RJ-70, acyclovir, α-interferon,recombinant sCD4, michellamines, calanolides, nonoxynol-9, gossypol andderivatives thereof, neuroamidase inhibitors, amantatadine, rimantadine,enfurtide, and gramicidin. If the composition is to be used to induce animmune response, it comprises an immune response-inducing amount of agriffithsin or conjugate thereof and can further comprise animmunoadjuvant, such as polyphosphazene polyelectrolyte. The griffithsinused in the composition, e.g., pharmaceutical composition, can beisolated and purified from nature or genetically engineered. Similarly,the griffithsin conjugate can be genetically engineered or chemicallycoupled.

The inventive compositions can be administered to a host, such as ahuman, so as to inhibit viral infection in a prophylactic or therapeuticmethod. The compositions of the invention are particularly useful ininhibiting the growth or replication of a virus, such as influenza virusor a retrovirus, in particular an influenza virus or an immunodeficiencyvirus, such as HIV, specifically HIV-1 and HIV-2, inhibiting infectivityof the virus, inhibiting the binding of virus to a host cell, and thelike. The compositions are useful in the therapeutic or prophylactictreatment of animals, such as humans, who are infected with a virus orwho are at risk for viral infection, respectively. The compositions alsocan be used to treat objects or materials, such as medical equipment,supplies, or fluids, including biological fluids, such as blood, bloodproducts and vaccine formulations, cells, tissues and organs, to removeor inactivate virus in an effort to prevent or treat viral infection ofan animal, such as a human. Such compositions also are useful to preventsexual transmission of viral infections, e.g., HIV, which is the primaryway in which the world's AIDS cases are contracted (Merson (1993),supra). Adherence of the inventive anti-viral polypeptide or conjugatethereof to a solid support, such as a filter, can be used in clinics toremove all or part of the viral content of a biological solution. Forexample, filters comprising the inventive anti-viral agents can be usedto treat blood supplies prior to transfusion to reduce the risk of viraltransmission. Such filters would find particular utility in clinicswherein risk of viral infection is high. It will be appreciated thattotal removal of the viral content of a biological solution is notrequired to achieve a beneficial effect. Removal of even a fraction ofvirus from a biological solution decreases the risk of infection of apatient.

Potential virucides used or being considered for use against sexualtransmission of HIV are very limited; present agents in this categoryinclude, for example, nonoxynol-9 (Bird, AIDS, 5: 791-796 (1991)),gossypol and derivatives (Polsky et al., Contraception, 39: 579-587(1989); Lin, Antimicrob. Agents Chemother, 33: 2149-2151 (1989); andRoyer, Pharmacol. Res, 24: 407-412 (1991)), and gramicidin (Bourinbair,Life Sci./Pharmacol. Lett, 54: PL5-9 (1994); and Bourinbair et al.,Contraception, 49: 131-137 (1994)). The method of prevention of sexualtransmission of viral infection, e.g., HIV infection, in accordance withthe invention comprises vaginal, rectal, oral, penile or other topicaltreatment with an anti-viral effective amount of a griffithsin and/orgriffithsin conjugate, alone or in combination with another anti-viralcompound as described herein.

In a novel approach to anti-HIV prophylaxis pursued under auspices ofthe U.S. National Institute of Allergy and Infectious Diseases (NIAID)(e.g., as conveyed by Painter, USA Today, Feb. 13, 1996), vaginalsuppository instillation of live cultures of lactobacilli was beingevaluated in a 900-woman study. This study was based especially uponobservations of anti-HIV effects of certain H₂O₂-producing lactobacilliin vitro (e.g., see published abstract by Hilier, from NIAID-sponsoredConference on “Advances in AIDS Vaccine Development,” Bethesda, Md.,Feb. 11-15, 1996). Lactobacilli readily populate the vagina, and indeedare a predominant bacterial population in most healthy women(Redondo-Lopez et al., Rev. Infect. Dis., 12: 856-872 (1990); Reid etal., Clin. Microbiol. Rev., 3: 335-344 (1990); Bruce and Reid, Can. J.Microbiol., 34: 339-343 (1988); Reu et al., J. Infect. Dis., 171:1237-1243 (1995); Hilier et al., Clin. Infect. Dis., 16 (Suppl 4):S273-S281; and Agnew et al., Sex. Transm. Dis., 22: 269-273 (1995)).Lactobacilli are also prominent, nonpathogenic inhabitants of other bodycavities such as the mouth, nasopharynx, upper and lowergastrointestinal tracts, and rectum.

It is well-established that lactobacilli can be readily transduced usingavailable genetic engineering techniques to incorporate a desiredforeign DNA coding sequence, and that such lactobacilli can be made toexpress a corresponding desired foreign protein (see, e.g., Hols et al.,Appl. and Environ. Microbiol., 60: 1401-1413 (1994)). Therefore, withinthe context of the present disclosure, it will be appreciated by oneskilled in the art that viable host cells containing a DNA sequence orvector of the invention, and expressing a polypeptide or fusion proteinof the invention, can be used directly as the delivery vehicle for agriffithsin or fusion protein thereof to the desired site(s) in vivo.Preferred host cells for such delivery of griffithsins or fusionproteins thereof directly to desired site(s), such as, for example, to aselected body cavity, can comprise bacteria or yeast. More specifically,such host cells can comprise suitably engineered strain(s) oflactobacilli, enterococci, or other common bacteria, such as E. coli,normal strains of which are known to commonly populate body cavities.More specifically yet, such host cells can comprise one or more selectednonpathogenic strains of lactobacilli, such as those described by Andreuet al. ((1995), supra), especially those having high adherenceproperties to epithelial cells, such as, for example, adherence tovaginal epithelial cells, and suitably transformed using the DNAsequences of the present invention.

As reviewed by McGroarty (FEMS Immunol. Med. Microbiol., 6: 251-264(1993)) the “probiotic” or direct therapeutic application of livebacteria, particularly bacteria that occur normally in nature, moreparticularly lactobacilli, for treatment or prophylaxis againstpathogenic bacterial or yeast infections of the urogenital tract, inparticular the female urogenital tract, is a well-established concept.Recently, the use of a conventional probiotic strategy, in particularthe use of live lactobacilli, to inhibit sexual transmission of HIV hasbeen suggested, based specifically upon the normal, endogenousproduction of virucidal levels of H₂O₂ and/or lactic acid and/or otherpotentially virucidal substances by certain normal strains oflactobacilli (e.g., Hilier (1996), supra). However, the inventive use ofnon-mammalian cells, particularly bacteria, more particularlylactobacilli, specifically engineered with a foreign gene, morespecifically a griffithsin gene, to express an anti-viral substance,more specifically a protein, and even more specifically a griffithsin,is heretofore unprecedented as a method of treatment of an animal,specifically a human, to prevent infection by a virus, specifically aretrovirus, more specifically HIV-1 or HIV-2.

Elmer et al. (JAMA, 275: 870-876 (1996)) have recently speculated that“genetic engineering offers the possibility of using microbes to deliverspecific actions or products to the colon or other mucosal surfaces . .. other fertile areas for future study include defining the mechanismsof action of various biotherapeutic agents with the possibility ofapplying genetic engineering to enhance activities.” Elmer et al.((1996), supra) further point out that the terms “probiotic” and“biotherapeutic agent” have been used in the literature to describemicroorganisms that have antagonistic activity toward pathogens in vivo;those authors more specifically prefer the term “biotherapeutic agent”to denote “microorganisms having specific therapeutic properties.”

In view of the present disclosure, one skilled in the art willappreciate that the invention teaches an entirely novel type of“probiotic” or “biotherapeutic” treatment using specifically engineeredstrains of microorganisms provided herein which do not occur in nature.Nonetheless, available teachings concerning selection of optimalmicrobial strains, in particular bacterial strains, for conventionalprobiotic or biotherapeutic applications can be employed in the contextof the invention. For example, selection of optimal lactobacillusstrains for genetic engineering, transformation, direct expression ofgriffithsins or conjugates thereof, and direct probiotic orbiotherapeutic applications, to treat or prevent viral (e.g., HIV)infection, can be based upon the same or similar criteria, such as thosedescribed by Elmer et al. ((1996), supra), typically used to selectnormal, endogenous or “nonengineered” bacterial strains for conventionalprobiotic or biotherapeutic therapy. Furthermore, the recommendationsand characteristics taught by McGroarty, particularly for selection ofoptimal lactobacillus strains for conventional probiotic use againstfemale urogenital infections, are pertinent to the present invention: “. . . lactobacilli chosen for incorporation into probiotic preparationsshould be easy and, if possible, inexpensive to cultivate . . . strainsshould be stable, retain viability following freeze-drying and, ofcourse, be non-pathogenic to the host . . . it is essential thatlactobacilli chosen for use in probiotic preparations should adhere wellto the vaginal epithelium . . . ideally, artificially implantedlactobacilli should adhere to the vaginal epithelium, integrate with theindigenous microorganisms present, and proliferate” (McGroarty (1993),supra). While McGroarty's teachings specifically address selections of“normal” lactobacillus strains for probiotic uses against pathogenicbacterial or yeast infections of the female urogenital tract, similarconsiderations will apply to the selection of optimal bacterial strainsfor genetic engineering and “probiotic” or “biotherapeutic” applicationagainst viral infections as particularly encompassed by the presentinvention.

Accordingly, the method of the invention for the prevention of sexualtransmission of viral infection, e.g., HIV infection, comprises vaginal,rectal, oral, penile, or other topical, insertional, or instillationaltreatment with an anti-viral effective amount of a griffithsin, agriffithsin conjugate or fusion protein, a matrix-anchored griffithsinor conjugate or fusion protein thereof, and/or viable host cellstransformed to express a griffithsin or conjugate or fusion proteinthereof, alone or in combination with one or more other anti-viralcompound (e.g., as described above). However, commensal organisms whichproduce griffithsin or a fragment, homolog, or conjugate thereof caninhibit viruses other than HIV. For example, commensal microorganismsthat produce the inventive polypeptide can be instilled in mucosaltissue at the site of influenza contact, such as nasal or oral mucosa,to inhibit influenza infection of a host.

Compositions for use in the prophylactic or therapeutic treatmentmethods of the invention comprise one or more griffithsin(s) orconjugate(s) or fusion protein(s) thereof, either one of which can bematrix-anchored, and desirably a carrier therefor, such as apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well-known to those who are skilled in the art, as aresuitable methods of administration. The choice of carrier will bedetermined in part by the particular griffithsin or conjugate or fusionprotein thereof, as well as by the particular method used to administerthe composition.

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routecan be used to administer a particular drug, a particular route canprovide a more immediate and more effective reaction than another route.For example, the anti-viral agent of the invention can be inhaled inmethods of prophylactically treating a subject for influenza infection.Delivery of the anti-viral agent to a location of initial viral contact,such as the nose or mouth, blocks the onset of infection. The anti-viralagent can be administered via subcutaneous injection. Alternatively, inacute or critical medical situations, the anti-viral agent can beadministered intravenously. In many cases of infection, a patientgenerates an immune response to a virus. However, the effects of theviral infection so severely compromise the health of the patient that aneffective immune response is not reached prior to death. Administrationof the anti-viral agent can prolong the life of the patient until apatient's natural immune defense clears the virus. Furthermore, oneskilled in the art will appreciate that the particular pharmaceuticalcarrier employed will depend, in part, upon the particular griffithsinor conjugate or fusion protein thereof employed, and the chosen route ofadministration. Accordingly, there is a wide variety of suitableformulations of the composition of the invention.

Formulations suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid, granules or freeze-dried cells; solutions orsuspensions in an aqueous liquid; and oil-in-water emulsions orwater-in-oil emulsions. Tablet forms can include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Suitable formulationsfor oral delivery can also be incorporated into synthetic and naturalpolymeric microspheres, or other means to protect the agents of thepresent invention from degradation within the gastrointestinal tract(see, for example, Wallace et al., Science, 260: 912-915 (1993)).

The anti-viral agent of the invention (e.g., griffithsin or conjugatesthereof), alone or in combination with other anti-viral compounds, canbe made into aerosol formulations or microparticulate powderformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

The anti-viral agent of the invention (e.g., griffithsin or conjugatesthereof), alone or in combinations with other anti-viral compounds orabsorption modulators, can be made into suitable formulations fortransdermal application and absorption, such as a patch (Wallace et al.(1993), supra). Transdermal electroporation or iontophoresis also can beused to promote and/or control the systemic delivery of the compoundsand/or compositions of the present invention through the skin (e.g., seeTheiss et al., Meth. Find. Exp. Clin. Pharmacol., 13: 353-359 (1991)).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, gels and the like containing, inaddition to the active ingredient, such as, for example, freeze-driedlactobacilli or live lactobacillus cultures genetically engineered todirectly produce a griffithsin or conjugate or fusion protein thereof ofthe present invention, such carriers as are known in the art. Topicaladministration is preferred for the prophylactic and therapeutictreatment of influenza viral infection, such as through the use of aninhaler, for example.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a griffithsin or conjugate orfusion protein thereof of the present invention, such carriers as areknown in the art to be appropriate. Similarly, the active ingredient canbe combined with a lubricant as a coating on a condom. Indeed,preferably, the active ingredient is applied to any contraceptivedevice, including, but not limited to, a condom, a diaphragm, a cervicalcap, a vaginal ring, and a sponge.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Formulations comprising a griffithsin or griffithsin conjugate suitablefor virucidal (e.g., HIV) sterilization of inanimate objects, such asmedical supplies or equipment, laboratory equipment and supplies,instruments, devices, and the like, can, for example, be selected oradapted as appropriate, by one skilled in the art, from any of theaforementioned compositions or formulations. Preferably, the griffithsinis produced by recombinant DNA technology. The griffithsin conjugate canbe produced by recombinant DNA technology or by chemical coupling of agriffithsin with an effector molecule as described above. Similarly,formulations suitable for ex vivo sterilization, inactivation, orremoval of virus, such as infectious virus, from a sample, such asblood, blood products, sperm, or other bodily products, such as a fluid,cells, a tissue or an organ, or any other solution, suspension,emulsion, vaccine formulation (such as in the removal of infectiousvirus), or any other material which can be administered to a patient ina medical procedure, can be selected or adapted as appropriate by oneskilled in the art, from any of the aforementioned compositions orformulations. However, suitable formulations for ex vivo sterilizationor inactivation or removal of virus from a sample or on an inanimateobject are by no means limited to any of the aforementioned formulationsor compositions. For example, such formulations or compositions cancomprise a functional griffithsin, such as that which is encoded by SEQID NO:3, or anti-viral fragment thereof, such as a fragment comprisingat least eight contiguous amino acids of SEQ ID NO:3, wherein the atleast eight contiguous amino acids bind to a virus, or a conjugate orfusion protein of either of the foregoing, attached to a solid supportmatrix, to facilitate contacting or binding infectious virus in a sampleor removing infectious virus from a sample as described above, e.g., abodily product such as a fluid, cells, a tissue or an organ from anorganism, in particular a mammal, such as a human, including, forexample, blood, a component of blood (e.g., plasma, blood cells, and thelike), or sperm. Preferably, the anti-viral polypeptide comprises SEQ IDNO:3. Also preferably, the at least eight contiguous amino acids bindgp120 of HIV, in particular infectious HIV. As a more specific example,such a formulation or composition can comprise a functional griffithsin,or conjugate or fusion protein thereof, attached to (e.g., coupled to orimmobilized on) a solid support matrix comprising magnetic beads, tofacilitate contacting, binding and removal of infectious virus, and toenable magnet-assisted removal of the virus from a sample as describedabove, e.g., a bodily product such as a fluid, cells, a tissue or anorgan, e.g., blood, a component of blood, or sperm. Alternatively, andalso preferably, the solid support matrix comprises a contraceptivedevice, such as a condom, a diaphragm, a cervical cap, a vaginal ring,or a sponge. The anti-viral agent also can be encapsulated or dispersedwithin a solid matrix, such as a vaginal ring or sponge. Methods forencapsulating biotherapeutics into, for example, biocompatible sustainedrelease devices, are known in the art.

As an even more specific illustration, such a composition (e.g., for exvivo) can comprise a functional (e.g., gp120-binding, HIV-inactivating)griffithsin, or conjugate or fusion protein thereof, attached to a solidsupport matrix, such as magnetic beads or a flow-through matrix, bymeans of an anti-griffithsin antibody or at least one effectorcomponent, which can be the same or different, such as polyethyleneglycol, albumin, or dextran. The conjugate can further comprise at leastone effector component, which can be the same or different, selectedfrom the group consisting of, for example, an immunological reagent anda toxin. A flow-through matrix would comprise, for instance, aconfiguration similar to an affinity column. The griffithsin can becovalently coupled to a solid support matrix via an anti-griffithsinantibody, described below. Methods of attaching an antibody to a solidsupport matrix are well-known in the art (see, for example, Harlow andLane. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory:Cold Spring Harbor, N.Y. (1988)). Alternatively, the solid supportmatrix, such as magnetic beads, can be coated with streptavidin, inwhich case the griffithsin or fragment thereof (which comprises at leasteight contiguous amino acids of SEQ ID NO:3 or SEQ ID NO:2), or aconjugate or fusion protein of either one, is biotinylated. The at leasteight contiguous amino acids of SEQ ID NO:2 desirably have anti-viralactivity and preferably bind gp120 of HIV, which preferably isinfectious. Preferably, the anti-viral polypeptide comprises SEQ ID NO:3or SEQ ID NO:2. Such a composition can be prepared, for example, bybiotinylating the griffithsin, or conjugate or fusion protein thereof,and then contacting the biotinylated protein or peptide with a(commercially available) solid support matrix, such as magnetic beads,coated with streptavidin. The use of biotinylation as a means to attacha desired biologically active protein or peptide to astreptavidin-coated support matrix, such as magnetic beads, iswell-known in the art.

One skilled in the art will appreciate that a suitable or appropriateformulation can be selected, adapted or developed based upon theparticular application at hand.

For ex vivo uses, such as virucidal treatments of inanimate objects ormaterials, blood or blood products, or tissues, the amount ofgriffithsin, conjugate thereof, fusion protein thereof, or compositionof any of the foregoing, to be employed should be sufficient that anyvirus or virus-producing cells present will be rendered noninfectious orwill be destroyed. For example, for HIV, this would require that thevirus and/or the virus-producing cells be exposed to concentrations ofgriffithsin in the range of 0.1-1000 nM. Similar considerations apply toin vivo applications. Therefore, the designation of “anti-viraleffective amount” is used generally to describe the amount of aparticular griffithsin, conjugate, fusion protein, or compositionthereof required for anti-viral efficacy in any given application.

In view of the above, the invention also provides a method of inhibitingprophylactically or therapeutically a viral infection of a host in whichan anti-viral effective amount of an above-described anti-viralpolypeptide, conjugate, or fusion protein is administered to the host.Upon administration of the anti-viral effective amount of the anti-viralpolypeptide, conjugate, or fusion protein, the viral infection isinhibited.

The invention additionally provides a method of prophylactically ortherapeutically inhibiting a viral infection of a host in which ananti-viral effective amount of a composition comprising an isolated andpurified anti-viral polypeptide, or anti-viral polypeptide conjugate orfusion protein, either one of which comprises at least eight contiguousamino acids of SEQ ID NO:3 having anti-viral activity, attached to orencapsulated within a solid support matrix is administered to the host.By “therapeutically” is meant that the host already has been infectedwith the virus. By “prophylactically” is meant that the host has not yetbeen infected with the virus but is at risk of being infected with thevirus. Prophylactic treatment is intended to encompass any degree ofinhibition of viral infection, including, but not limited to, completeinhibition, as one of ordinary skill in the art will readily appreciatethat any degree in inhibition of viral infection is advantageous.Preferably, the inventive active agent is administered before viralinfection or immediately upon determination of viral infection and iscontinuously administered until the virus is undetectable. The methodoptionally further comprises the prior, simultaneous or subsequentadministration, by the same route or a different route, of an antiviralagent or another agent that is efficacious in inhibiting the viralinfection. Upon administration of the anti-viral effective amount of thecomposition, the viral infection is inhibited. Preferably, the solidsupport matrix is a contraceptive device, such as a condom, diaphragm,cervical cap, vaginal ring, or sponge. In an alternative embodiment, asolid support matrix can be surgically implanted and later removed.

For in vivo uses, the dose of a griffithsin, or conjugate or compositionthereof, administered to an animal, particularly a human, in the contextof the invention should be sufficient to effect a prophylactic ortherapeutic response in the individual over a reasonable time frame. Thedose used to achieve a desired anti-viral concentration in vivo (e.g.,0.1-1000 nM) will be determined by the potency of the particulargriffithsin or conjugate employed, the severity of the disease state ofinfected individuals, as well as, in the case of systemicadministration, the body weight and age of the infected individual. Thesize of the dose also will be determined by the existence of any adverseside effects that may accompany the particular griffithsin, or conjugateor composition thereof, employed. It is always desirable, wheneverpossible, to keep adverse side effects to a minimum.

The invention also provides a method of removing virus, such asinfectious virus, from a sample. The method comprises contacting thesample with a composition comprising an isolated and purified anti-viralpolypeptide or conjugate or fusion protein thereof, comprising at leasteight contiguous amino acids of SEQ ID NO:3 (or SEQ ID NO: 2). The atleast eight contiguous amino acids desirably have anti-viral activityand bind to the virus and the anti-viral polypeptide (or conjugate orfusion protein of either of the foregoing) is attached to a solidsupport matrix, such as a magnetic bead. “Attached” is used herein torefer to attachment to (or coupling to) and immobilization in or on asolid support matrix. While any means of attachment can be used,preferably, attachment is by covalent bonds. The method furthercomprises separating the sample and the composition by any suitablemeans, whereupon the virus, such as infectious virus, is removed fromthe sample. Preferably, the anti-viral polypeptide comprises SEQ ID NO:3(or SEQ ID NO:2). In one embodiment, the anti-viral polypeptide isconjugated with an anti-griffithsin antibody or at least one effectorcomponent, which can be the same or different, selected frompolyethylene glycol, dextran and albumin, in which case the anti-viralpolypeptide is desirably attached to the solid support matrix through atleast one effector component. The anti-viral polypeptide can be furtherconjugated with at least one effector component, which can be the sameor different, selected from the group consisting of an immunologicalreagent and a toxin. In another embodiment, the solid support matrix iscoated with streptavidin and the anti-viral polypeptide is biotinylated.Through biotin, the biotinylated anti-viral polypeptide is attached tothe streptavidin-coated solid support matrix. Other types of means, asare known in the art, can be used to attach a functional griffithsin(i.e., an anti-viral polypeptide or conjugate as described above) to asolid support matrix, such as a magnetic bead, in which case contactwith a magnet is used to separate the sample and the composition.Similarly, other types of solid support matrices can be used, such as amatrix comprising a porous surface or membrane, over or through which asample is flowed or percolated, thereby selectively entrapping orremoving infectious virus from the sample. The choice of solid supportmatrix, means of attachment of the functional griffithsin to the solidsupport matrix, and means of separating the sample and thematrix-anchored griffithsin will depend, in part, on the sample (e.g.,fluid vs. tissue) and the virus to be removed. It is expected that theuse of a selected coupling molecule can confer certain desiredproperties to a matrix, comprising a functional griffithsin coupledtherewith, that may have particularly advantageous properties in a givensituation. Preferably, the sample is blood, a component of blood, sperm,cells, tissue or an organ. Also, preferably the sample is a vaccineformulation, in which case the virus that is removed is infectious, suchas HIV, although HIV, in particular infectious HIV, can be removed fromother samples in accordance with this method.

For instance, the skilled practitioner might select a poly(ethyleneglycol) molecule for attaching a functional griffithsin to a solidsupport matrix, thereby to provide a matrix-anchored griffithsin,wherein the griffithsin is attached to the matrix by a longer “tether”than would be feasible or possible for other attachment methods, such asbiotinylation/streptavidin coupling. A griffithsin coupled by apoly(ethylene glycol) “tether” to a solid support matrix (such asmagnetic beads, porous surface or membrane, and the like) can permitoptimal exposure of a binding surface, epitope, hydrophobic orelectrophilic focus, and/or the like, on a functional griffithsin in amanner that, in a given situation and/or for a particular virus,facilitates the binding and/or inactivation of the virus. A preferredsolid support matrix is a magnetic bead such that separation of thesample and the composition is effected by a magnet. In a preferredembodiment of the method, the at least eight contiguous amino acids bindgp120 of HIV and HIV is removed from the sample.

Similarly, other types of solid support matrices can be used, such as amatrix comprising a porous surface or membrane, over or through which asample is flowed or percolated, thereby selectively inhibitinginfectious virus (e.g., HIV or influenza) in the sample. The choice ofsolid support matrix, means of attachment of the functional griffithsinto the solid support matrix, and means of separating the sample and thematrix-anchored griffithsin will depend, in part, on the sample (e.g.,fluid vs. tissue) and the virus to be inhibited. It is expected that theuse of a selected coupling molecule can confer certain desiredproperties to a matrix, comprising a functional griffithsin coupledtherewith, that may have particularly advantageous properties in a givensituation.

The methods described herein also have utility in real time ex vivoinhibition of virus or virus infected cells in a bodily fluid, such asblood, e.g., in the treatment of viral infection, or in the inhibitionof virus in blood or a component of blood, e.g., for transfusion, in theinhibition or prevention of viral infection. Such methods also havepotential utility in dialysis, such as kidney dialysis, and ininhibiting virus in sperm obtained from a donor for in vitro and in vivofertilization. The methods also have applicability in the context oftissue and organ transplantations.

In summary, a griffithsin attached to a solid support matrix, such as amagnetic bead, can be used to remove virus, in particular infectiousvirus, including immunodeficiency virus, such as HIV, e.g., HIV-1 orHIV-2, from a sample, such as a sample comprising both infectious andnoninfectious virus. The inventive method also can be used to removeviral glycoprotein presenting cells, e.g., infected cells that have, forexample, gp120 on their surfaces, from a sample.

The invention, therefore, further provides a composition comprisingnaturally-occurring, non-infectious virus, such as a compositionproduced as described above. The composition can further comprise acarrier, such as a biologically or pharmaceutically acceptable carrier,and an immuno-adjuvant. Preferably, the noninfectious virus is aninfluenza or an immunodeficiency virus, such as HIV, e.g., HIV-1 orHIV-2. Alternatively, and also preferably, the noninfectious virus isFIV. A composition comprising only naturally-occurring, non-infectiousvirus has many applications in research and the prophylactic treatmentof a viral infection. In terms of prophylactic treatment of a viralinfection, the skilled artisan will appreciate the need to eliminatecompletely all infectious virus from the composition. If desired,further treatment of the composition comprising non-infectious particleswith virus-inactivating chemicals, such as imines or psoralens, and/orpressure or heat inactivation, will further the non-infectious nature ofthe composition. For example, an immune response-inducing amount of theinventive composition can be administered to an animal at risk for aviral infection in order to induce an immune response. The skilledartisan will appreciate that such a composition is a significantimprovement over previously disclosed compositions in that the virus isnon-infectious and naturally-occurring. Thus, there is no risk ofinadvertent infection, greater doses can be administered in comparisonto compositions comprising infectious viral particles, and thesubsequent immune response will assuredly be directed to antigenspresent on naturally-occurring virus. The composition comprisingnaturally-occurring, non-infectious virus can be administered in anymanner appropriate to induce an immune response. Preferably, the virusis administered, for example, intramuscularly, mucosally, intravenously,subcutaneously, or topically. Preferably, the composition comprisesnaturally-occurring, non-infectious human immunodeficiency viruscomprising gp120.

The composition comprising naturally-occurring, non-infectious virus canbe combined with various carriers, adjuvants, diluents or otheranti-viral therapeutics, if desired. Appropriate carriers include, forexample, ovalbumin, albumin, globulins, hemocyanins, and the like.Adjuvants or immuno-adjuvants are incorporated in most cases tostimulate further the immune system. Any physiologically appropriateadjuvant can be used. Suitable adjuvants for inclusion in the inventivecomposition include, for example, aluminum hydroxide, beryllium sulfate,silica, kaolin, carbon, bacterial endotoxin, saponin, and the like.

Thus, the invention also provides a method of inducing an immuneresponse to a virus in an animal. The method comprises administering tothe animal an immune response-inducing amount of a compositioncomprising naturally-occurring, non-infectious virus as described above.

The appropriate dose of a composition comprising naturally-occurring,non-infectious virus required to induce an immune response to the virusin an animal is dependent on numerous factors, such as size of theanimal and immune competency. The amount of composition administeredshould be sufficient to induce a humoral and/or cellular immuneresponse. The amount of non-infectious virus in a particular compositioncan be determined using routine methods in the art, such as the CoulterHIV p24 antigen assay (Coulter Corp., Hialeah, Fla.). Any suitable doseof a composition comprising non-infectious virus is appropriate so longas an immune response is induced, desirably without the appearance ofharmful side effects to the host. In this regard, compositionscomprising from about 10¹ to about 10⁵ particles, preferably from about10² to about 10⁴ particles, most preferably about 10³ particles, aresuitable for inducing an immune response.

One of ordinary skill can determine the effectiveness of the compositionto induce an immune response using routine methods known in the art.Cell-mediated response can be determined by employing, for example, avirus antigen-stimulated T-cell proliferation assay. The presence of ahumoral immune response can be determined, for instance, with the EnzymeLinked Immunosorbent Assay (ELISA). The skilled artisan will appreciatethat there are numerous other suitable assays for evaluating inductionof an immune response. To the extent that a dose is inadequate to inducean appropriate immune response, “booster” administrations cansubsequently be administered in order to prompt a more effective immuneresponse.

In terms of administration of the inventive anti-viral agents orconjugates thereof, the dosage can be in unit dosage form, such as atablet or capsule. The term “unit dosage form” as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of agriffithsin or conjugate thereof, alone or in combination with otheranti-viral agents, calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier, or vehicle.

The specifications for the unit dosage forms of the invention depend onthe particular griffithsin, or conjugate or composition thereof,employed and the effect to be achieved, as well as the pharmacodynamicsassociated with each griffithsin, or conjugate or composition thereof,in the host. The dose administered should be an “anti-viral effectiveamount” or an amount necessary to achieve an “effective level” in theindividual patient.

Since the “effective level” is used as the preferred endpoint fordosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective level” can be defined, for example, as theblood or tissue level (e.g., 0.1-1000 nM) desired in the patient thatcorresponds to a concentration of one or more griffithsin or conjugatethereof, which inhibits a virus, such as HIV, in an assay known topredict for clinical anti-viral activity of chemical compounds andbiological agents. The “effective level” for agents of the inventionalso can vary when the griffithsin, or conjugate or composition thereof,is used in combination with AZT or other known anti-viral compounds orcombinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectiveconcentration” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectiveconcentration” of the compounds of the invention by a direct (e.g.,analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it can bedesirable to utilize a “mega-dosing” regimen, wherein a large dose ofthe griffithsin or conjugate thereof is administered, time is allowedfor the drug to act, and then a suitable reagent is administered to theindividual to inactivate the drug.

The pharmaceutical composition can contain other pharmaceuticals, inconjunction with the griffithsin or conjugate thereof, when used totherapeutically treat a viral infection, such as an influenza infectionor an HIV infection which results in AIDS. Representative examples ofthese additional pharmaceuticals include anti-viral compounds,virucides, immunomodulators, immunostimulants, antibiotics andabsorption enhancers. Exemplary anti-viral compounds include cyanovirin,AZT, ddI, ddC, gancylclovir, fluorinated dideoxynucleosides,nonnucleoside analog compounds, such as nevirapine (Shih et al., PNAS,88: 9878-9882 (1991)), TIBO derivatives, such as R82913 (White et al.,Anti-viral Res., 16: 257-266 (1991)), BI-RJ-70 (Merigan, Am. J. Med., 90(Suppl. 4A):8S-17S (1991)), michellamines (Boyd et al., J. Med. Chem.,37: 1740-1745 (1994)) and calanolides Kashman et al., J. Med. Chem., 35:2735-2743 (1992)), nonoxynol-9, gossypol and derivatives, gramicidin(Bourinbair et al. (1994), supra), neuraminidase inhibitors, amantadine,enfurtide, and the like. Exemplary immunomodulators and immunostimulantsinclude various interleukins, sCD4, cytokines, antibody preparations,blood transfusions, and cell transfusions. Exemplary antibiotics includeantifungal agents, antibacterial agents, and anti-Pneutnoeystitis carniiagents. Exemplary absorption enhancers include bile salts and othersurfactants, saponins, cyclodextrins, and phospholipids (Davis (1992),supra).

Administration of a griffithsin or conjugate or fusion protein thereofwith other anti-retroviral agents and particularly with known RTinhibitors, such as ddC, AZT, ddI, ddA, or other inhibitors that actagainst other HIV proteins, such as anti-TAT agents, is expected toinhibit most or all replicative stages of the viral life cycle. Thedosages of ddC and AZT used in AIDS or ARC patients have been published.A virustatic range of ddC is generally between 0.05 μM to 1.0 μM. Arange of about 0.005-0.25 mg/kg body weight is virustatic in mostpatients. The preliminary dose ranges for oral administration aresomewhat broader, for example 0.001 to 0.25 mg/kg given in one or moredoses at intervals of 2, 4, 6, 8, 12, etc. hours. Currently, 0.01 mg/kgbody weight ddC given every 8 hrs is preferred. When given in combinedtherapy, the other anti-viral compound, for example, can be given at thesame time as the griffithsin or conjugate thereof or the dosing can bestaggered as desired. The two drugs also can be combined in acomposition. Doses of each can be less when used in combination thanwhen either is used alone.

It will also be appreciated by one skilled in the art that a DNAsequence of a griffithsin or conjugate thereof of the invention can beinserted ex vivo into mammalian cells previously removed from a givenanimal, in particular a human, host. Such cells can be employed toexpress the corresponding griffithsin or conjugate or fusion protein invivo after reintroduction into the host. Feasibility of such atherapeutic strategy to deliver a therapeutic amount of an agent inclose proximity to the desired target cells and pathogens, i.e., virus,more particularly retrovirus, specifically HIV and its envelopeglycoprotein gp120, has been demonstrated in studies with cellsengineered ex vivo to express sCD4 (Morgan et al. (1994), supra). It isalso possible that, as an alternative to ex vivo insertion of the DNAsequences of the invention, such sequences can be inserted into cellsdirectly in vivo, such as by use of an appropriate viral vector. Suchcells transfected in vivo are expected to produce anti-viral amounts ofgriffithsin or a conjugate or fusion protein thereof directly in vivo.

Given the present disclosure, it will be additionally appreciated that aDNA sequence corresponding to a griffithsin or conjugate thereof can beinserted into suitable nonmammalian host cells, and that such host cellswill express therapeutic or prophylactic amounts of a griffithsin orconjugate or fusion protein thereof directly in vivo within a desiredbody compartment of an animal, in particular a human. Example 5illustrates the transformation and expression of effective virucidalamounts of a griffithsin in a non-mammalian cell, more specifically abacterial cell. In a preferred embodiment of the invention, a method offemale-controllable prophylaxis against HIV infection comprises theintravaginal administration and/or establishment of, in a female human,a persistent intravaginal population of lactobacilli that have beentransformed with a coding sequence of the invention to produce, over aprolonged time, effective virucidal levels of a griffithsin or conjugatethereof, directly on or within the vaginal and/or cervical and/oruterine mucosa. It is noteworthy that both the World Health Organization(WHO), as well as the U.S. National Institute of Allergy and InfectiousDiseases, have pointed to the need for development of female-controlledtopical microbicides, suitable for blocking the transmission of HIV, asan urgent global priority (Lange et al., Lancet, 341: 1356 (1993);Fauci, NIAID News, Apr. 27, 1995). A composition comprising theinventive anti-viral agent and a solid-support matrix is particularlyuseful in this regard, particularly when the solid-support matrix is acontraceptive device, such as a condom, a diaphragm, a cervical cap, avaginal ring, or a sponge. In another embodiment, a colony of commensalorganisms transduced with the nucleic acid of the invention andproducing the inventive anti-viral agent is applied to mucosal tissueassociated with the onset of influenza infection, such as respiratory ororal mucosal.

The invention also provides antibodies directed to the polypeptides ofthe invention. The availability of antibodies to any given protein ishighly advantageous, as it provides the basis for a wide variety ofqualitative and quantitative analytical methods, separation andpurification methods, and other useful applications directed to thesubject polypeptides. Accordingly, given the present disclosure and thepolypeptides of the invention, it will be readily apparent to oneskilled in the art that antibodies, in particular antibodiesspecifically binding to a polypeptide of the invention, can be preparedusing well-established methodologies (e.g., such as the methodologiesdescribed in detail by Harlow and Lane, in Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988), pp.1-725). Such antibodies can comprise both polyclonal and monoclonalantibodies. Furthermore, such antibodies can be obtained and employedeither in solution-phase or coupled to a desired solid-phase matrix,such as magnetic beads or a flow through matrix. Having in hand suchantibodies as provided by the invention, one skilled in the art willfurther appreciate that such antibodies, in conjunction withwell-established procedures (e.g., such as described by Harlow and Lane(1988), supra) comprise useful methods for the detection,quantification, or purification of a griffithsin, conjugate thereof, orhost cell transformed to produce a griffithsin or conjugate or fusionprotein thereof. Example 6 further illustrates an antibody thatspecifically binds to a griffithsin. Accordingly, the invention furtherprovides a composition comprising an anti-griffithsin antibody bound tothe anti-viral agent of the invention, preferably an anti-viralpolypeptide comprising at least eight contiguous amino acids of SEQ IDNO:3.

Matrix-anchored anti-griffithsin antibodies also can be used in a methodto remove virus in a sample. Preferably, the antibody binds to anepitope of an anti-viral polypeptide of SEQ ID NO:2 or SEQ ID NO:3.Preferably, the matrix is a solid support matrix, such as a magneticbead or a flow-through matrix. If the solid support matrix to which theanti-griffithsin antibody is attached comprises magnetic beads, removalof the antibody-griffithsin-virus complex can be readily accomplishedusing a magnet.

In view of the above, the invention provides a method of removing virusfrom a sample. The method comprises (a) contacting the sample with acomposition comprising an isolated and purified anti-viral polypeptideor conjugate or fusion protein thereof, wherein (i) the anti-viralpolypeptide comprises at least eight contiguous amino acids of SEQ IDNO: 3, and (ii) the at least eight contiguous amino acids bind to thevirus, and (b) contacting the sample with an anti-griffithsin antibodyattached to a solid support matrix, whereupon the anti-griffithsinantibody binds to the anti-viral polypeptide or conjugate or fusionprotein thereof to which is bound the virus, and (c) separating thesolid support matrix from the sample, whereupon the virus is removedfrom the sample. Preferably, the anti-viral polypeptide comprises SEQ IDNO:3. Desirably, the virus that is removed is infectious, such as HIV.The sample can be blood, a component of blood, sperm, cells, tissue oran organ.

The antibody for use in the aforementioned method is an antibody thatbinds to a polypeptide comprising at least eight contiguous amino acidsof SEQ ID NO:3, and, which polypeptide can bind to and inactivate avirus. The antibody can be coupled to the solid support matrix usingsimilar methods and with similar considerations as described above forattaching a griffithsin to a solid support matrix. For example, couplingmethods and molecules employed to attach an anti-griffithsin antibody toa solid support matrix, such as magnetic beads or a flow-through matrix,can employ biotin/streptavidin coupling or coupling through molecules,such as polyethylene glycol, albumin or dextran. Also analogously, itcan be shown that, after such coupling, the matrix-anchoredanti-griffithsin antibody retains its ability to bind to a polypeptidecomprising at least eight contiguous amino acids of SEQ ID NO:3, whichpolypeptide can bind to and inactivate a virus.

The invention also provides an anti-griffithsin antibody that isanti-idiotypic in respect to a viral glycoprotein, such as gp120, i.e.,has an internal image of gp120 of a primate immunodeficiency virus.Preferably, the antibody can compete with gp120 of a primateimmunodeficiency virus for binding to a griffithsin. In this regard, theprimary immunodeficiency virus preferably is HIV-1 or HIV-2 and thegriffithsin preferably consists essentially of SEQ ID NO:2 or SEQ IDNO:3. Anti-idiotypic antibodies can be generated in accordance withmethods known in the art (see, for example, Benjamin, in Immunology: ashort course, Wiley-Liss, N Y (1996), pp. 436-437; Kuby, in Immunology,3rd ed., Freeman, N.Y. (1997), pp. 455-456; Greenspan et al., FASEB J.,7: 437-443 (1993); and Poskitt, Vaccine, 9: 792-796 (1991)). Such ananti-idiotypic (in respect to gp120) anti-griffithsin antibody is usefulin a method of inhibiting infection of an animal with a virus asprovided herein.

In view of the above, a griffithsin can be administered to an animal,the animal generates anti-griffithsin antibodies, among which areantibodies that have an internal image of a viral glycoprotein, such asgp120. In accordance with well-known methods, polyclonal or monoclonalantibodies can be obtained, isolated, and selected. Selection of ananti-griffithsin antibody that has an internal image of gp120 can bebased upon competition between the anti-griffithsin antibody and gp120for binding to a griffithsin, or upon the ability of theanti-griffithsin antibody to bind to a free griffithsin as opposed to agriffithsin bound to gp120. Such an anti-griffithsin antibody can beadministered to an animal to inhibit a viral infection in accordancewith methods provided herein. Although nonhuman anti-idiotypicantibodies, such as an anti-griffithsin antibody that has an internalimage of gp120 and, therefore, is anti-idiotypic to gp120, are provinguseful as vaccine antigens in humans, their favorable properties might,in certain instances, be further enhanced and/or their adverseproperties further diminished, through “humanization” strategies, suchas those recently reviewed by Vaughan (Nature Biotech., 16: 535-539(1998)). Alternatively, a griffithsin can be directly administered to ananimal to inhibit a viral infection in accordance with methods providedherein such that the treated animal, itself, generates ananti-griffithsin antibody that has an internal image of gp120. Theproduction of anti-idiotypic antibodies, such as anti-griffithsinantibody that has an internal image of gp120 and, therefore, isanti-idiotypic to gp120, in an animal to be treated is known as“anti-idiotype induction therapy,” and is described by Madiyalakan etal. (Hybridoma, 14: 199-203 (1995)), for example.

In view of the above, the invention enables another method of inhibitinginfection of an animal, such as a mammal, in particular a human, with avirus. The method comprises administering to the animal ananti-griffithsin antibody, or a composition comprising same, in anamount sufficient to induce in the animal an immune response to thevirus, whereupon the infection of the animal with the virus isinhibited. Preferably, the anti-griffithsin antibody has an internalimage of a viral glycoprotein, such as gp120 of an immunodeficiencyvirus with which the animal can be infected, such as a primateimmunodeficiency virus. Preferably, the antibody can compete with, forexample, gp120 of a primate immunodeficiency virus for binding to agriffithsin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV-2 and the griffithsin preferably consistsessentially of SEQ ID NO:3 or SEQ ID NO:2. The method can furthercomprise the administration of an immunostimulant.

Also enabled by the invention is yet another method of inhibitinginfection of an animal, such as a mammal, in particular a human, with avirus. The method comprises administering to the animal a griffithsin,which binds a viral glycoprotein, such as gp120 of an immunodeficiencyvirus with which the animal can be infected, in an amount sufficient toinduce in the animal an anti-griffithsin antibody in an amountsufficient to induce an immune response to a virus sufficient to inhibitinfection of the animal with the virus. Preferably, the anti-griffithsinantibody has an internal image of gp120 of an immunodeficiency viruswith which the animal can be infected, such as a primateimmunodeficiency virus. Preferably, the antibody can compete with gp120of a primate immunodeficiency virus for binding to a griffithsin. Inthis regard, the primate immunodeficiency virus preferably is HIV-1 orHIV-2 and the griffithsin preferably consists essentially of SEQ ID NO:2or SEQ ID NO:3.

With respect to the above methods, sufficient amounts can be determinedin accordance with methods known in the art. Similarly, the sufficiencyof an immune response in the inhibition of a viral infection in ananimal also can be assessed in accordance with methods known in the art.

Either one of the above methods can further comprise concurrent, pre- orpost-treatment with an adjuvant to enhance the immune response, such asthe prior, simultaneous or subsequent administration, by the same or adifferent route, of an antiviral agent or another agent that isefficacious in inducing an immune response to the virus, such as animmunostimulant. See, for example, Harlow et al. (1988), supra.

The inventive griffithsins, conjugates, host cells, antibodies,compositions and methods are further described in the context of thefollowing examples. These examples serve to illustrate further thepresent invention and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

This example illustrates a method of isolating and purifying griffithsinfrom Griffithsin sp. and elucidating the griffithsin amino acidsequence.

Anti-HIV bioassay guided fractionation was used to track the isolationof the griffithsin polypeptide. In brief, the cellular mass fromGriffithsia sp. was harvested by filtration, freeze-dried, and extractedfirst with H₂O followed by (1:1) MeOH-CH₂Cl₂. Individual aliquots of theorganic and aqueous extracts were tested for cytoprotective propertiesin the NCI primary anti-HIV screen (Weislow et al. J. Natl. CancerInst., 81: 577-586 (1989)). Only the H₂O extract showed anti-HIVactivity.

A freeze-dried aqueous extract (10 g) was brought to a concentration of50 mg/ml by addition of DDH₂O and maintained on ice. Crystallineammonium sulfate (Sigma, St. Louis, Mo.; molecular biology grade) wasadded to the solution such that the final concentration of the mixturewas 75% saturation. The mixture was allowed to precipitate on ice overnight, and was then centrifuged at 3000 rpm for 50 min. The resultingpellets were set aside. The supernatant was brought to 1 M ammoniumsulfate followed by another round of precipitation and centrifugation.The pellets from the second centrifugation were saved, and the resultingsupernatant was filtered using a 0.22 μm filter and subjected tohydrophobic interaction chromatography. A BioCad workstation (PerseptiveBiosystems) was used for the following column chromatographies. Theprotein solution from the centrifugation and filtration steps wasinjected onto a Poros PE column (10×100 mm, Perseptive Biosystems)pre-equilibrated with a starting buffer of 50 mM sodium phosphate, 1.5 Mammonium sulfate, pH 7.5. The column was eluted at a flow rate of 15ml/min over the following gradient: (1) 7 column volumes (CV, equal to7.85 ml) of the starting buffer; (2) 1.5-0 M ammonium sulfate over 2 CV;(3) 0 M ammonium sulfate for 15 CV. The eluate was monitored for bothconductivity and absorbance (280 nm). Ammonium sulfate was added to thevoid fraction possessing anti-HIV activity to bring the finalconcentration to 75% saturation. The mixture was allowed to precipitateon ice overnight, and was then centrifuged at 3000 rpm for 50 min.DDH₂O-resuspended pellets were first concentrated using a 10 kDamolecular weight limit membrane, dialyzed against 0.02% sodium azide,and then brought up to a concentration of 25 mM Tris-HCl, pH 8.5. Theresulting protein solution was injected onto a Poros HQ anion exchangecolumn (10×100 mm, Perseptive Biosystems) pre-equilibrated with astarting buffer of 25 mM Tris-HCl, pH 8.5. The column was eluted at aflow rate of 15 ml/min using the following gradient: (1) 5 CV of thestarting buffer; (2) 0-1 M sodium chloride over 20 CV; (3) 1 M sodiumchloride for 5 CV. The eluate was monitored for absorbance (280 nm).Active fractions from the HQ column were concentrated and desalted usinga 10 kDa molecular weight limit membrane and subjected to a Bio-RP C4reverse phase column (4.6×100 mm, Covance, Princeton, N.J.) and elutedat a flow rate of 4 ml/min using the following gradient: (1) 10 CV ofthe starting buffer of 5% acetonitrile in H₂O; (2) 5-95% acetonitrile inH₂O over 2.5 CV; (3) 95% acetonitrile in H₂O for 5 CV. The eluate wasmonitored for absorbance (280 nm), and the active fraction was pooled,lyophilized, and resuspended in phosphate-buffered saline (PBS), pH 7.4.The protein solution was injected onto a G3000PW gel permeation column(21.5×600 mm, TosoHaas, Montgomeryville, Pa.) and eluted with PBS, pH7.4, at a flow rate of 5 ml/min.

Molecular mass and purity (>99%) of griffithsin were confirmed byElectrospray ionization mass spectrometry (ESI-MS), and the proteinconcentrations were determined by amino acid analysis. Native molecularweight was determined by calibrating standard proteins (albumin (68kDa), cytochrome c (12.5 kDa), and aprotinin (6.5 kDa)) by theirretention time (as measured by absorbance at 280 nm) and comparing theresulting calibration curve to the retention time of the active protein.Amino acid analysis was accomplished using a Beckman Model 6300Automated Amino Acid Analyzer according to manufacturer protocols.N-terminal amino acid sequencing was performed using an AppliedBiosystems Model 4774A Sequencer according to manufacturer protocols.Matrix-assisted laser desorption ionization-time of flight massspectroscopy (MALDI-TOF MS) was performed using a Kratos Kompact MaldiIII instrument (Shimadzu, Columbia, Md.) operated in a linear mode usingsinapinic acid as a matrix and trypsin as an external standard. ESI-MSwas performed with a JEOL SX102 equipped with an Analytica electrospraysource. The spectrometer was calibrated using a lysozyme standard(molecular weight=14305.2) prior to each analysis. Samples were injectedinto the source in a 1:1 solution of hexafluorosopropanol and 2% aceticacid. The masses reported were averages calculated from the variouscharged states observed.

Griffithsin was subjected to digestion with cyanogen bromide (CNBr) anda variety of endoproteinases (Lys-C, Arg-C, and Asp-N) permanufacturer's instructions. The cleaved peptide products were purifiedby reversed-phase HPLC using a gradient of 0.05% aqueous trifluoroaceticacid for 20 min, then increasing to 60% acetonitrile in 0.05% aqueoustrifluoroacetic acid over 100 min. Amino acid sequences were determinedby sequential Edman degradation using an Applied Biosystems Model 494sequencer according to the protocols of the manufacturer, and the massesof cleaved peptides were analyzed by MALDI-TOF mass spectrometer. Theamino acid sequence of the native griffithsin polypeptide is set forthas SEQ ID NO:3.

In summary, the preliminary analysis of the crude aqueous extract ofalgae Griffithsia sp. in the NCI's primary in vitro anti-HIV screeningassay (Weislow et al., supra) identified a protein that bound solublegp120. The process described herein is illustrated in FIG. 1. Anti-HIVbioassay-guided fractionation of the aqueous resulted in the isolationof griffithsin. The aqueous extract was subjected to ammonium sulfateprecipitation, hydrophobic interaction chromatography, anion exchangechromatography, reversed-phase chromatography, and size exclusionchromatography to produce a homogeneous protein fraction. SDS-PAGEanalysis showed a single protein band with a relative molecular mass ofapproximately 13 kDa, named griffithsin. Purified griffithsin exhibiteda single band by immunoblotting with anti-griffithsin polyclonalantibodies. The amino acid sequence of the purified griffithsin wasestablished by N-terminal Edman degradation of the intact protein and byN-terminal sequencing of peptide fragments cleaved by CNBr and a varietyof endopeptidases (Lys-C, Arg-C, and Asp-N) followed by reversed phasepurification and MALDI-TOF mass spectrometric analysis. The entire 121amino acid sequence was established except for a single amino acid atposition 31, which does not match any of the common amino acids.Electrospray ionization mass spectrometric analysis of isolatedgriffithsin showed a molecular ion with m/z 12,770.05, and thecalculated value for the deduced amino acid sequence without amino acidat position 31 was m/z 12619.00. It was deduced that the molecular massof the amino acid at position 31 was 151.05. The amino acid analysis ofgriffithsin also agreed with the deduced primary sequence. These datafully support the proposed primary amino acid sequence of griffithsin. Asearch of the BLAST database (Altschul et al., Nucleic Acids Res, 25(17), 3389-3402 (1997)) for identification of protein sequencesimilarities did not reveal any homologies of greater than eightcontiguous amino acids nor >30% total sequence homology betweengriffithsin and any amino acid sequences of known proteins ortranscription products of known nucleotide sequences, including theanti-HIV proteins cyanovirin-N and scytovirin.

Example 2

This example demonstrates the synthesis of griffithsin genes. Themethods described herein are illustrated in FIG. 2.

The chemically deduced amino acid sequence of griffithsin wasback-translated to elucidate the corresponding DNA coding sequence.Since amino acid residue 31 of native griffithsin did not appear to beone of the twenty common amino acids, alanine was substituted in thisposition (SEQ ID NO:2). In order to facilitate initial production andpurification of recombinant griffithsin, a commercial expression vectorpET-26b(+), from Novagen, Inc., Madison, Wis., for which reagents wereavailable for affinity purification and detection, was selected.Appropriate restriction sites for ligation to pET-26b(+), and a stopcodon, were included in the DNA sequence. SEQ ID NO:1 is an example of aDNA sequence encoding a synthetic griffithsin gene. A flowchartillustrating a method of synthesizing of a griffithsin gene is shown inFIG. 2.

A griffithsin-encoding DNA sequence was synthesized as 13 overlapping,complementary oligonucleotides and assembled to form the double-strandedcoding sequence. Oligonucleotide elements of the synthetic DNA codingsequence were synthesized using a nucleic acid synthesizer (model 394,Applied Biosystems Inc., Foster City, Calif.). The purified 13oligonucleotides were individually treated with T4 polynucleotidekinase, and 1 nM quantities of each were pooled and boiled for 10minutes to ensure denaturation. The temperature of the mixture was thenreduced to 70° C. for annealing of the complementary strands for 15minutes, and further reduced to 60° C. for 15 minutes. The reaction wascooled on ice and T4 DNA ligase (2,000 units) additional ligase bufferwas added to the reaction. Ligation of the oligonucleotides wasperformed with T4 DNA ligase overnight at 16° C. The resulting DNA wasrecovered and purified from the reaction buffer by phenol:chloroformextraction, ethanol precipitation, and further washing with ethanol.

The purified, double-stranded synthetic DNA was then used as a templatein a polymerase chain reaction (PCR). One μl of the DNA solutionobtained after purification of the ligation reaction mixture was used asa template. Thermal cycling was performed using a Perkin-Elmerinstrument. “Pfu” thermostable DNA polymerase, restriction enzymes, T4DNA ligase, and polynucleotide kinase were obtained from Stratagene, LaJolla, Calif. Pfu polymerase was selected for this application becauseof its claimed superiority in fidelity compared to the usual Taq enzyme.The PCR reaction product was run on a 2% agarose gel in TAE buffer. The465 base pair DNA construct was cut from the gel and purified. Thepurified DNA, which was digested with Nde I and Xho I restrictionenzymes, was then ligated into the multicloning site of the pet-26b(+)vector.

E. coli were transfected with the generated pET-26b(+)-construct, andrecombinant clones were identified by analysis of restriction digests ofplasmid DNA. Sequence analysis of one of these selected clones indicatedthat three bases deviated from the intended coding sequence. These“mutations,” which presumably arose during the PCR amplification of thesynthetic template, were corrected by a site-directed mutagenesis kitfrom Stratagene, La Jolla, Calif. The repair was confirmed by DNAsequence analysis.

For preparation of a DNA sequence encoding a griffithsin polypeptidetagged with a penta-His peptide at the C-terminal end of griffithsin(e.g., SEQ ID NO:4), the aforementioned recombinant griffithsinconstruct was subjected to site-directed mutagenesis to eliminate stopcodons located between the griffithsin coding sequence and the penta-Hispeptide coding sequence using a site-directed mutagenesis kit fromStratagene, La Jolla, Calif. A pair of mutagenic oligonucleotide primerswere synthesized, which included portions of the codons encoding thegriffithsin polypeptide and penta-His peptide, but lacked the stopcodons. Annealing of these mutagenic primers with the template DNA andextension by DNA polymerase resulted in the generation of a DNAconstruct encoding a fusion protein comprising the griffithsin aminoacid sequence linked to a penta-His peptide tag. DNA sequencing verifiedthe presence of the intended sequence.

Example 3

This example demonstrates the expression of an N-terminalHis-tagged-griffithsin gene.

A recombinant griffithsin protein and a C-terminal, His-taggedgriffithsin protein encoded by the nucleic acids of Example 2 did notefficiently translocate to the periplasmic fraction of E. coli followingprotein expression. In addition, the majority of the produced proteinsaccumulated in the inclusion bodies of E. coli without the cleavage of apelB signal sequence located at the N-terminus of the griffithsinprotein. Thus, steps were taken to express griffithsin in the cytosolicfraction of E. coli.

The pET-26b(+)-griffithsin DNA construct was used as a template PCRusing a pair of appropriate primers. The PCR product was designed tohave a “penta-His” peptide and thrombin recognition site at theN-terminal end of the griffithsin polypeptide, providing for productionof a N-terminal, His-tagged-griffithsin fusion protein. The PCR reactionproduct was purified from an agarose gel. The purified DNA, which wasdigested with Nco I and Xho I restriction enzymes, was ligated into theexpression vector pET-28a(+) vector (Novagen, Inc., Madison, Wis.).

E. coli (strain BL21 (DE3)) were transfected with the pET-28a(+) vectorcontaining the nucleic acid coding sequence for theHis-tagged-griffithsin fusion protein (see SEQ ID NO:4). Selected cloneswere seeded into small-scale shake flasks containing LB growth mediumwith 30 μg/ml kanamycin and expanded by incubation at 37° C.Larger-scale Erlenmeyer flasks (0.5-3.0 liters) were then seeded. Theculture was allowed to grow to a density of 0.5-0.7 OD₆₀₀ units. Theexpression of the His-tagged-griffithsin fusion protein was induced byadding IPTG to a final concentration of 1 mM and continuing incubationat 37° C. for 3-6 hrs. Bacteria were harvested by centrifugation, andthe soluble fraction was obtained using BugBuster™ reagent and Benzonasenuclease (Novagen, Inc., Madison, Wis.). Crude soluble fractions showedboth anti-HIV activity and presence of a His-tagged-griffithsin fusionprotein by Western-blotting. In addition, the His-tagged-griffithsinprotein accumulated in the inclusion bodies of E. coli. A flowchartillustrating a method of expressing and purifying recombinantHis-tagged-griffithsin is shown in FIG. 3.

The purity (˜98%) of recombinant His-tagged griffithsin was confirmed bySDS-PAGE on 16% Tricine gel stained by Coomassie Blue staining. Theprotein showed the expected molecular mass for griffithsin (i.e., 14.6kDa). Protein concentrations were determined based on extinctioncoefficient at 280 nm of the protein. Approximately 1.6 mg ofrecombinant His-tagged griffithsin was purified from 1 L of E. coliculture. The purified protein demonstrated gp120-binding and anti-viralactivity equivalent to that of native griffithsin.

This example illustrates a method of producing recombinant griffithsin,which displays physical and functional properties similar, if notidentical, to that of natural griffithsin.

Example 4

This example describes a method of purifying a recombinantHis-tagged-griffithsin protein.

Using an immobilized metal affinity chromatography set-up includingNi-NTA agarose (QIAGEN Inc., Valencia, Calif.), a His-tagged-griffithsinfusion protein (as described in Example 3) was purified.

The soluble fraction described in Example 3 was loaded onto 20 mlgravity columns containing affinity matrix. The columns were washedextensively with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mMimidazole, pH 8.0) to remove contaminating proteins. Since His-taggedgriffithsin cannot compete for binding sites on the Ni-NTA resin if theimidazole concentration is increased to 100-250 mM, the His-taggedgriffithsin protein was eluted by applying elution buffer (50 mMNaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0) through the column.Column fractions and wash volumes were monitored by Western-blotanalysis using Penta-His™ antibody (QIAGEN Inc., Valencia, Calif.) oranti-griffithsin antibody. Fractions containing the purified His-taggedgriffithsin protein were pooled, dialyzed extensively against distilledwater, and lyophilized.

Potent cytoprotective and anti-replicative activities of both naturaland His-tagged recombinant griffithsin were observed using the HIV-1RFstrain of HIB in CEM-SS cells. Both the natural and recombinantgriffithsin polypeptides demonstrated a concentration-dependentinhibition of virus-induced cell killing. Griffithsin treatment alsoresulted in concomitant decreases in supernatant reverse transcriptaseand viral core antigen, p24. Mid-to-high picomolar concentrations ofgriffithsin exhibited comparably potent activity against all of therepresentative T-tropic laboratory strains and primary isolates as wellas M-tropic primary isolates. In the antiviral assays, there was littleor no evidence of direct cytotoxicity of griffithsin to the uninfectedcontrol cells at the highest tested concentrations of griffithsin (78.3to 783 nM). Griffithsin-pretreated uninfected CEM-SS cells retainednormal susceptibility to HIV infection after the removal of griffithsin.In contrast, infectivity of cell-free virus was abolished afterpretreatment and removal of griffithsin. These results indicate thatgriffithsin is a virucide. Cocultivation of uninfected and chronicallyinfected CEM-SS with griffithsin resulted in concentration-dependentinhibition of cell-cell fusion. Additional binding and fusion inhibitionassay using β-gal indicator cells showed similar results. Griffithsininhibited fusion of CD4 β-gal cells with HL ⅔ cells and also inhibitedcell-free HIV-1IIIB fusion and infection of β-gal cells in aconcentration-dependent manner.

Example 5

This example illustrates the anti-HIV activity of natural griffithsinpolypeptide and His-tagged griffithsin polypeptide.

Pure proteins were initially evaluated for antiviral activity using anXTT-tetrazolium anti-HIV assay described previously (Boyd, in Aids,Etiology, Diagnosis, Treatment And Prevention (1988), supra; Gustafsonet al., J. Med. Chem., 35: 1978-1986 (1992); Weislow (1989), supra;Gulakowski (1991), supra). A CEM-SS human lymphocytic target cell linewas used in all assays maintained in RPMI 1650 medium (Gibco, GrandIsland, N.Y.), without phenol red, supplemented with 5% fetal bovineserum, 2 mM L-Glutamine, and 50 mg/ml Gentamicin (complete medium).

Exponentially growing cells were pelleted and resuspended at aconcentration of 2.0×10⁵ cells/ml in complete medium. The Haitianvariant of HIV, HTLV-III_(RF) (3.54×10⁶ SFU/ml), was used throughout.Frozen virus stock solutions were thawed immediately before use andresuspended in complete medium to yield 1.2×10⁵SFU/ml. The appropriateamounts of the pure proteins for anti-HIV evaluations were dissolved inH₂O-DMSO (3:1), then diluted in complete medium to the desired initialconcentration. All serial drug dilutions, reagent additions, andplate-to-plate transfers were carried out with an automated Biomek 1000Workstation (Beckman Instruments, Palo Alto, Calif.).

FIG. 4 summarizes the observed antiviral activities of nativegriffithsin from Griffithsia sp. (FIG. 4 a) and recombinantHis-tagged-fusion griffithsin (FIG. 4 b). Effects of a range ofconcentrations of native griffithsin and HIS-tagged-griffithsin uponCEM-SS cells infected with HIV-1, as determined after 6 days in cultureis illustrated in FIG. 6. Data points represent the percent of therespective uninfected, nondrug-treated control values. The twogriffithsin polypeptides demonstrated potent anti-HIV activity with anEC₅₀ in the low nanomolar range and no significant evidence of directcytotoxicity to the host cells at the highest tested concentrations (upto 1 mM).

Example 6

This example demonstrates that HIV viral envelope gp120 is the principaltarget for griffithsin.

To determine the affinity of griffithsin for a series of proteinstandards, 100 ng each of gp160, gp120, gp41, sCD4, bovine IgG, α-acidglycoprotein, and aprotinin were subjected to ELISA as previouslydescribed (Bokesch et al., Biochemistry, 42: 2578-2584 (2003)). Briefly,the protein standards were bound to a 96-well plate, which was rinsedwith PBST (three times) and blocked with BSA. Between each step of theprotocol, the plate was rinsed with PBST (three times). The proteinstandards were incubated with griffithsin (100 ng/well), followed byincubation with a 1:500 dilution of an anti-griffithsin rabbitpolyclonal antibody preparation. Griffithsin bound to the proteinstandards was detected by adding goat-anti-rabbit antibodies conjugatedto alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis,Ind.). Upon addition of alkaline phosphatase substrate buffer,absorbance was measured at 405 nm for each well. Glycosylation-dependentbinding of griffithsin to gp120 was examined using an ELISA as above,with glycosylated and nonglycosylated gp120 (HIV-1_(SF2) gp120) added tothe 96-well plate and incubated with serial dilutions of griffithsin.

Griffithsin was tested for its ability to bind HIV envelopeglycoproteins. Evidence for direct interaction of griffithsin withgp120, gp160, and to a lesser degree, gp41 was obtained from ELISAexperiments (FIG. 5 a). There was little or no detectable interactionbetween griffithsin and cCD4 or other reference proteins, includingbovine IgG, α-acid glycoprotein, and aprotinin. An additional ELISAexperiment showed that binding of griffithsin to sgp120 is bothconcentration-dependent and glycosylation-dependent (FIG. 5 b).

To undertake preliminary mapping studies to define griffithsin-bindingsite on the gp120, we evaluated the effect of griffithsin on thereactivity of soluble CD4 (sCD4), cyanovirin-N, and a panel ofmonoclonal antibodies (mAb) with soluble gp120 (sgp120) in an ELISAformat assay. These studies demonstrated that griffithsin interferedstrongly with recognition of sgp120 by the mAbs 48d and 2G12.Griffithsin moderately interfered with sCD4 and mAb IgG1b12 binding tosgp120. Griffithsin had little or no effect on the recognition of sgp120by mAbs that recognize the C1 region (or V3 loop), and the mAb 17b.However, additional studies demonstrated that pretreatment of sgp120with sCD4 and the mAbs IgG 1b12, 48d, and 2G12 did not block subsequentbinding of griffithsin to sgp120. Cyanovirin-N interfered strongly withthe recognition of sgp120 by griffithsin. On the other hand, griffithsinpretreatment of sgp120 did not block subsequent binding of cyanovirin-Nto sgp120.

Since griffithsin inhibited viral entry, we compared matched control andgriffithsin-treated sgp120 preparations in a flow cytometricsgp120/CD4-expressing cell binding assay to determine whethergriffithsin inhibits viral attachment or subsequent fusion events. TheCEM-SS cell line expresses CD4, as demonstrated by the binding of targetcells with both anti-Leu3a and anti-OKT4 monoclonal antibodies. Afterincubation of CEM-SS cells with sgp120, the cells were stained byanti-gp120 mAb-FITC. A concomitant decrease in the availability of theLeu3a epitope (i.e., the gp120-binding site on target cells) wasobserved. In other words, the sgp120 bound to the gp120 binding site onthe target cells. As expected, little change in the staining specificfor the OKT4 epitope (i.e., a non-gp120 binding site) was observed.These results are consistent with sgp120 binding of CD4 on the targetcells. Pretreatment of sgp120 with griffithsin substantially recoveredthe availability of the Leu3a epitope, indicating that griffithsincompletely blocked CD4-dependent sgp120 binding. However, overall sgp120binding showed two peaks in the flow cytometry data whengriffithsin-treated sgp120 was added to the cells. The decreased signalsuggests inhibition of sgp120 binding to CD4 by griffithsin, which wasconsistent with the recovery of the availability of the Leu3a epitope.The increased signal suggests that the griffithsin/sgp120 complex alsonon-specifically bound to target cells.

This example demonstrates that griffithsin binds to a region of gp120that recognizes CD4 on host cells.

Example 7

This example illustrates the broad-range anti-HIV activity ofgriffithsin.

Anti-viral assays used to study the activities of laboratory strains andprimary isolates of virus have been previously published (Buckheit etal., Antiviral Res., 21: 247-265 (1993)). The low passage HIV-1pediatric isolate ROJO was derived as previously described (Buckheit etal., AIDS Res. Hum. Retroviruses, 10: 1497-1506 (1994)). Peripheralblood mononuclear cells (PBMC) and macrophages were isolated fromhepatitis and HIV sero-negative donors following Ficoll-Hypaquecentrifugation as described elsewhere (Gartner and Popovic, Techniquesin HIV Research, Aldovini, A. and Walker, B., eds., Stockton Press, NewYork (1994) pp. 59-63). Mean EC₅₀ values were determined fromconcentration-response curves from eight dilutions of griffithsin(triplicate wells/concentration); assays for HIV-1 RF/CEM-SS employedXTT-tetrazolium; HIV-1 ROJO were tested in human PBMC cultures bysupernatant reverse transcriptase activity; HIV-1 Ba-L and ADA weretested in human primary macrophage cultures by p24 ELISA assay. Standarderrors averaged less than 10% of the respective means. The results ofthis study are summarized in Table 1 below.

TABLE 1 Virus Target Cell Tropism EC₅₀ (nM) HIV-1 Laboratory Strain RFCEM-SS T 0.043 HIV Primary Isolates ROJO PBMC T 0.63 ADA Macrophage M0.50 Ba-L Macrophage M 0.098

The results show that griffithsin is potently active (sub-nanomolar EC₅₀values) against a broad range of HIV isolates including T-tropic viruses(utilizing CCR5 as a co-receptor) and M-tropic viruses (utilizing CXCR4as a co-receptor). This picomolar level of activity is more potent thanthat described for most of the current anti-HIV agents utilized intherapy or in development, including the entry inhibitors cyanovirin-Nand Enfurtide®. The data also show that griffithsin is effective atinhibiting infection by both laboratory-adapted strains and, moreimportantly, primary clinical isolates of HIV (e.g., ROJO, ADA, andBa-L). Finally, the results indicate that griffithsin is activeregardless of the cell type used in the assay, having potent activitywhether the cells were T-lymphocytes (CEM-SS), PBMCs, or macrophages.Griffithsin did not show any toxicity against any of the cell lines evenat concentrations 1000-fold higher than the EC₅₀ values.

Example 8

This example describes the production of anti-griffithsin polyclonalantibodies. A flow diagram illustrating a method of producinganti-griffithsin antibodies is provided in FIG. 6.

A New Zealand white rabbit was immunized with 100 μg of griffithsin inFreund's complete adjuvant. Booster injections of 50 μg of griffithsinin Freund's incomplete adjuvant were administered on days 13, 29, 51,64, 100, and 195. On days 7, 21, 42, 63, 78, and 112, 10 mL of blood wasremoved from the rabbit. On day 112 the rabbit was sacrificed and bledout. The IgG fraction of the immune sera of the rabbit was isolated byprotein-A Sepharose affinity chromatography (Bio-Rad, Hercules, Calif.)according to the manufacturer's instructions. Reactivity of thepolyclonal antibodies for griffithsin was demonstrated by immunoblot andELISA studies with 1:500 to 1:3000 dilution of the rabbit immunoglobulinfractions.

For immunoblotting, samples were transferred to PVDF membranes followingSDS-PAGE according to standard procedures. The membranes were incubatedfor 1 hour with anti-griffithsin polyclonal antibodies, washed threetimes with PBS containing 0.05% Tween 20 (PBST), and then treated withgoat anti-rabbit IgG antibodies conjugated to horseradish peroxidase(Sigma, St. Louis, Mo.). After three washes with PBST, bound antibodieswere visualized by incubating membranes in a solution of 0.05%3,3′-diaminobenzidine and 0.003% H₂O₂.

The IgG fraction of rabbit polyclonal anti-griffithsin antibodies werepurified after the final boost and animal sacrifice by using protein-ASepharose chromatography on the 57 mL of rabbit serum collected.Following purification, 78 mL of purified anti-griffithsin IgGs wereproduced. The final concentration of protein was 335 micrograms/mL for atotal yield of 27.3 mg of anti-griffithsin IgG. To analyze thespecificity of the resulting antibody preparation, Western blot analysiswas performed and resulted in the clear determination of specificity andavidity for griffithsin by the purified antibodies. A 1:250 dilution ofthe purified antibodies clearly visualized only the griffithsin from amixture of griffithsin and other proteins. The response to griffithsinby the anti-griffithsin antibodies was also shown to beconcentration-dependent.

Example 9

This example illustrates the anti-influenza virus activity ofgriffithsin.

All examined influenza viruses were passaged in Madin Darby caninekidney (MDCK) cells to prepare viral stocks. MDCK cells (from ATCC,Manassas, Va.) were grown in antibiotic-free minimum essential medium(MEM) with non-essential amino acids (Gibco, Long Island, N.Y.)containing 5% fetal bovine serum (FBS, HyClone Laboratories, Logan,Utah) and 0.1% NaHCO3. Test medium consisted of MEM with 0.18% NaHCO3,10 units/mL trypsin, 1 μg of ethylenediaminetetraacetate (EDTA) per ml,and 50 μg gentamicin/mL.

Inhibition of virus-induced cytopathic effect (CPE) as determined byvisual (microscopic) examination of infected cells and confirmed byincrease in neutral red (NR) dye uptake into infected cells was used asan indicator of griffithsin antiviral activity. The CPE inhibitionmethod was reported previously by Smee et al. (Antiviral Res., 5:251-259 (2001)). Seven concentrations of griffithsin were screened forantiviral activity against each virus in 96-well flat-bottomedmicroplates of cells. The griffithsin protein was added 5-10 minutesprior to addition of virus to the cells. The concentration of viruscorrespond to approximately 50% infection of cells in culture (CCID₅₀)per well. The virus challenge dose equals a multiplicity of infection ofapproximately 0.001 infectious particles per cell. The reactionproceeded at 37° C. for 72 hr. To perform the NR uptake assay forconfirmation of antiviral activity, dye (0.34% concentration in medium)was added to the plates used to obtain visual scores of CPE. After 2hours, color intensity of the dye absorbed by and subsequently elutedfrom the cells was determined by the method of Finter et al., J. Gen.Virol., 5, 419-427 (1969) using a computerized EL-309 microplateautoreader (Bio-Tek Instruments, Winooski, Vt.). Antiviral activity wasexpressed as the 50% effective (virus-inhibitory) concentration (EC₅₀value) determined by plotting griffithsin concentration versus percentinhibition on semi-logarithmic graph paper. Cytotoxicity of compoundswas assessed in parallel with the antiviral determinations in the samemicroplates, except in the absence of virus. From these, 50% cytotoxicendpoints (IC₅₀ values) were determined. The results of this study aresummarized in Table 2.

TABLE 2 Influenza Virus Strain EC₅₀ (μg/ml) Beijing/262/95 (H1N1) 0.07Texas/36/91 (H1N1) 0.06 Los Angeles/2/87 (H3N2) 0.037 Panama/2007/99(H3N2) 0.006 Shandong/09/93 (H3N2) 0.018 Sydney/5/97 (H3N2) 0.016Washington/05/96 (H3N2) 0.016

Similar to the results with HIV, griffithsin was found to be potentlyactive against a wide spectrum of influenza A viruses. These virusesincluded both H1N1 strains and H3N2 strains of influenza, which isespecially significant in light of the fact that the highly virulentFijian strain of influenza A that afflicted the United States in2003/2004 was also a H3N2 strain. Griffithsin was not found to be toxicto the MDCK cell line utilized for these experiments, even when thecells were exposed to a high dose of griffithsin (10 micrograms/mL).

Example 10

This example describes a method of producing recombinant griffithsin.

Recombinant expression of His-tagged griffithsin in E. coli wasoptimized using a fermenter in combination with an auto-induction media.A seed culture was grown in LB media containing 30 μg/ml kanamycin in ashaker flask at 37° C. and 150 rpm for 17 hours. In addition, afermenter containing an auto-induction media was inoculated with theseed culture. The ratio of auto-induction media to seed culture wasapproximately 50:1. The culture was grown at 37° C. for 24 hours. Thefinal culture density was approximately 8.6 OD₆₀₀ units. The finalculture was harvested by centrifugation, and the soluble fraction wasobtained as described above.

Crude soluble fractions contained His-tagged-griffithsin fusion protein,which was detected by Western-blotting with anti-griffithsin polyclonalantibodies. The ratio of soluble:insoluble protein at approximately 15kDa was 50:50. The ratio indicates that more griffithsin protein wasproduced in soluble fraction in this fermentation procedure comparedwith protein expression achieved using a shaker flask procedure. Inaddition, the fermentation procedure provided approximately 30-foldhigher quantities of griffithsin protein than the shaker flaskprocedure. Approximately 50 mg of purified recombinant griffithsin wasisolated from 1 L of the fermentation. The purified protein existed as ahomodimer and demonstrated gp120 binding and anti-viral activityequivalent to that of native griffithsin.

The results of this example confirm a method of producing recombinant,anti-viral griffithsin protein.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   -   Birren et al., Cold Spring Harbor Laboratory Press, Cold Spring        Harbor, N.Y. (1997),    -   Birren et al., Genome Analysis: A Laboratory Manual Series,        Volume 2, Detecting Genes, Cold Spring Harbor Laboratory Press,        Cold Spring Harbor, N.Y. (1998),    -   Birren et al., Genome Analysis: A Laboratory Manual Series,        Volume 3, Cloning Systems, Cold Spring Harbor Laboratory Press,        Cold Spring Harbor, N.Y. (1999),    -   Birren et al., Genome Analysis: A Laboratory Manual Series,        Volume 4, Mapping Genomes, Cold Spring Harbor Laboratory Press,        Cold Spring Harbor, N.Y. (1999),    -   Harlow et al., Antibodies: A Laboratory Manual, Cold Spring        Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988),    -   Harlow et al., Using Antibodies: A Laboratory Manual, Cold        Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999),        and    -   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd        edition, Cold Spring Harbor Laboratory Press, Cold Spring        Harbor, N.Y. (1989).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of inhibiting a viral infection of a host, which methodcomprises administering to the host an effective amount of an anti-viralpolypeptide or anti-viral polypeptide conjugate comprising SEQ ID NO:3or a variant thereof, the variation consisting of rendering one or moreof amino acid residues 45, 60, 71, or 104 of SEQ ID NO:3 glycosylationresistant, wherein the anti-viral polypeptide or anti-viral polypeptideconjugate has anti-viral activity, whereupon the viral infection isinhibited.
 2. The method of claim 1, wherein the viral infection is aninfluenza viral infection.
 3. The method of claim 2, wherein theanti-viral polypeptide or anti-viral polypeptide conjugate isadministered topically to the host.
 4. The method of claim 3, whereinthe anti-viral polypeptide or anti-viral polypeptide conjugate isadministered topically to the respiratory system.
 5. The method of claim4, wherein the anti-viral polypeptide or anti-viral polypeptideconjugate is administered as an aerosol or microparticulate powder. 6.The method of claim 1, wherein the viral infection is a retroviralinfection.
 7. The method of claim 6, wherein the retroviral infection isa human immunodeficiency viral infection.
 8. A method of inhibiting aviral infection of a host, which method comprises administering to thehost an effective amount of an anti-viral polypeptide or anti-viralpolypeptide conjugate comprising SEQ ID NO:3, whereupon the viralinfection is inhibited.
 9. The method of claim 8, wherein the viralinfection is an influenza viral infection.
 10. The method of claim 9,wherein the anti-viral polypeptide or anti-viral polypeptide conjugateis administered topically to the host.
 11. The method of claim 10,wherein the anti-viral polypeptide or anti-viral polypeptide conjugateis administered topically to the respiratory system.
 12. The method ofclaim 11, wherein the anti-viral polypeptide or anti-viral polypeptideconjugate is administered as an aerosol or microparticulate powder. 13.The method of claim 8, wherein the viral infection is a retroviralinfection.
 14. The method of claim 13, wherein the retroviral infectionis a human immunodeficiency viral infection.
 15. A method of inhibitinga viral infection of a host, which method comprises administering to thehost an effective amount of an anti-viral polypeptide or anti-viralpolypeptide conjugate comprising SEQ ID NO:2, whereupon the viralinfection is inhibited.
 16. The method of claim 15, wherein the viralinfection is an influenza viral infection.
 17. The method of claim 16,wherein the anti-viral polypeptide or anti-viral polypeptide conjugateis administered topically to the host.
 18. The method of claim 17,wherein the anti-viral polypeptide or anti-viral polypeptide conjugateis administered topically to the respiratory system.
 19. The method ofclaim 18, wherein the anti-viral polypeptide or anti-viral polypeptideconjugate is administered as an aerosol or microparticulate powder. 20.The method of claim 15, wherein the viral infection is a retroviralinfection.
 21. The method of claim 20, wherein the retroviral infectionis a human immunodeficiency viral infection.